Compositions and methods for treating or preventing inflammatory diseases

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DESCRIPTION

Use of anti-microtubule agents to treat inflammatory diseases of the respiratory tract.

Technical field

The present invention relates to the use of an anti-microtubule agent to prepare a medicament for treating or preventing inflammatory diseases of the respiratory tract.

Background of the invention

Inflammatory diseases, both chronic and acute in nature, represent a substantial problem in the healthcare industry. Briefly, chronic inflammation is considered to be inflammation of a long duration (weeks or months) in which active inflammation, tissue destruction, and attempts to heal take place simultaneously (Robbins Pathological Basis of Disease by RS Cotran, V Kumar and SL Robbins, WB Saunders Co., p. 75, 1989). Although chronic inflammation may be followed by an acute inflammatory episode, it may also begin as an insidious procedure that progresses over time, for example as a consequence of a persistent infection (e.g. tuberculosis, syphilis, fungal infection) causing a reaction of delayed hypersensitivity, prolonged exposure to endogenous (e.g. elevated plasma lipids) or exogenous (e.g. silica, asbestos, cigarette tar or surgical satures), or autoimmune reactions against the body's own tissues (for example, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, or psoriasis). Thus, chronic inflammatory diseases include many common medical conditions such as rheumatoid arthritis, restenosis, psoriasis, multiple sclerosis, surgical adhesions, tuberculosis, and chronic inflammatory lung diseases (eg, asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps. and pulmonary fibrosis).

Inflammatory lung diseases

Chronic inflammatory lung diseases, including, for example, asthma, pneumoconiosis, chronic obstructive pulmonary disease, nasal polyps, and pulmonary fibrosis, affect many people around the world. These diseases are usually characterized by an invasive inflammatory procedure and a thickening of the affected tissues.

For example, nasal polyps are characterized by a thickened lining of the nose. Polyps can occur in respiratory diseases such as asthma, cystic fibrosis, primary ciliary dyskinesia, and autoimmune deficiencies. Nasal polyps are believed to develop as a manifestation of chronic inflammatory procedures that affect the upper respiratory tract. It has been found in 36% of patients with aspirin intolerance, 7% of them with asthma, 0.1% in children, and approximately 20% in those with cystic fibrosis. Other conditions associated with nasal polyps are Churg-Strauss syndrome, allergic fungal sinusitis, and ciliary dyskinetic syndrome and Young's syndrome. Approximately 40% of patients with surgical polypectomies relapsed (Settipane, Allergy Asthma Proc. 17 (5): 231-236, 1996).

The main symptoms of nasal polyposis are nasal obstruction and disturbance of the sense of smell. The goals of medical treatment for nasal polyposis are to (1) eliminate nasal polyps and rhinitis symptoms, (2) re-establish nasal breathing and smell, and (3) prevent relapse. Nasal passage occlusion by a few large polyps can be treated by a simple polypectomy to help the patient breathe through the nose. The goal of surgery is to restore the physiological properties of the nose and make the airways as free of polyps as possible and allow the infected sinuses to drain. However, recurrent nasal polyposis is one of the most common unsolved problems in clinical rhinology. Complementary medical treatment for polyposis is always necessary as surgery cannot treat the inflammatory component of mucosal disease. Topical corticosteroids are the most widely used treatment to shrink polyps and prevent relapse after surgery. Steroids reduce rhinitis, improve nasal breathing, reduce the size of polyps and decrease the rate of relapse, but they have a negligible effect on the sense of smell and on any sinus pathology. However, the use of steroids in polyposis is associated with infectious complications that require antibiotics. Other drugs for the treatment of nasal polyposis include H1 receptor antagonists (eg, azelastine-HCl) and anti-diuretics (eg, furosimide). These treatments are not always effective and relapse rates are still very high. Current medical treatment for nasal polyposis uses corticosteroids to alleviate the symptoms of the disease, but they have no action against the underlying pathology of the disease. Furthermore, disease relapse or resistance to steroid therapy has been observed in patients with nasal polyps.

Summary of the invention

Briefly stated, the present invention provides the use of an anti-microtubule agent for preparing a medicament for treating or preventing inflammatory diseases, which comprises delivering an anti-microtubule agent to a site of inflammation. Representative examples of these agents include taxanes (eg, paclitaxel and docetaxel), campothecin, eleutherobine, sarcodictyin, epothilones A and B, discodermolide, deuterium oxide (D2O), hexylene glycol (2-methyl-2,4-pentanediol), tubercidin (7-de-sazaadenosine), LY29081 (2-amino-4- (3-pyridyl) -4H-naphtho

ES 2 207 451 T3 (1,2-b) pyran-3-carbonitrile), aluminum fluoride, ethylene glycol bis- (succinimidylsuccinate), glycine ethyl ester, anti-idiotypic monoclonal antibodies, microtubule array-promoting protein (protein taxol-type, TALP), hypotonic-induced cell swelling (190 mosmol / l), insulin (10 nmol / l) or glutamine (10 nmol / l), binding to dynein, gibberlin, XCHO1 (kinesin-like protein), lysophosphatidic acid, lithium ion, plant cell wall components (e.g. poly-L-lysine and extensin), glycerol buffers, Triton X-100 microtubule stabilizing buffer, microtubule-associated proteins (e.g. MAP2, MAP4, tau, large tau, ensconsin, elongation factor 1-alpha (EF-1a) and E-MAP-115), cellular entities (for example, histone H1, myelin basic protein, and kinetochore), endogenous microtubule structures (for example, axonemal structures, plugs and GTP auctions), Stable tubule polypeptide alone (eg, STOP145 and STOP220 and mitotic force tension, as well as any analogs and derivatives of any of the foregoing. Within other embodiments, the anti-microtubule agent is formulated to further comprise a polymer.

Within certain embodiments of the invention, the anti-microtubule agents used according to the invention can be formulated together with other compounds or compositions such as, for example, an ointment, cream, lotion, gel, spray or the like. Within certain embodiments, the compound or composition can act as a carrier, which can be polymeric or non-polymeric. Representative examples of polymeric carriers include poly (ethylene vinyl acetate), copolymers of lactic acid and glycolic acid, poly (caprolactone), poly (lactic acid), copolymers of poly (lactic acid) and poly (caprolactone), gelatin, hyaluronic acid, collagen and albumen matrices. Representative examples of other suitable carriers include, but are not limited to ethanol, mixtures of ethanol and glycols (eg, ethylene glycol or propylene glycol); mixtures of ethanol and isopropyl myristate or ethanol, isopropyl myristate and water (eg, 55: 5:40); mixtures of ethanol and eineol or d-limonene (with or without water); glycols (eg ethylene glycol or propylene glycol) and glycol mixtures such as propylene glycol and water, phosphatidyl glycerol, dioleoyl phosphatidyl glycerol. Transcutol^® or terpinolene; mixtures of isopropyl myristate and 1-hexyl-2-pyrrolidone, N-dodecyl-2-piperidinone or 1-hexyl-2-pyrrolidone.

Within still other aspects, the anti-microtubule agent may be formulated to be contained or adapted to be delivered by a surgical or medical device or implant such as, for example, surgical shunts, sutures, internal catheters, prostheses, and the like. .

These and other aspects described will become more apparent upon reference to the following detailed description and accompanying drawings. In addition, various references are set forth below that describe certain procedures, devices, or compositions in more detail and are therefore incorporated by reference in their entirety.

Brief description of the drawings

Figure 1A is a graph showing the chemiluminescence response of neutrophils (5 x 10⁶ cells / ml) to plasma opsonized CPPD crystals (50 mg / ml). The effect of paclitaxel (also referred to as "taxol") at (O) without paclitaxel, (·) 4.5 µM, (Δ) 14 µM, (A) (28 µM, (or) 46 µM; n = 3. Figure 1B is a graph showing the concentration dependence over time of inhibition by paclitaxel of plasma opsonized CPPD crystals induced neutrophil chemiluminescence. Figure 1C is a graph showing the effect of aluminum fluoride on zymosan-induced neutrophil activation, as measured by chemiluminescence. Figure 1D is a graph showing the effect of glycine ethyl ester on opsonized zymosan-induced neutrophil activation as measured by chemiluminescence. Figure 1E is a graph showing the effect of LY290181 on opsonized zymosan-induced neutrophil chemiluminescence.

Figure 2 is a graph showing lysozyme release from neutrophils (5 x 10⁶/ ml) in response to plasma opsonized CPPD crystals (50 mg / ml). The effect of paclitaxel at (O) without paclitaxel, (·) 28 μM, (Δ) control (cells alone), (A) controls (cells and paclitaxel at 28 μM), n = 3.

Figure 3A is a graph showing superoxide anion production by neutrophils (5 x 10⁶ cells / ml) in response to plasma opsonized CPPD crystals (50 mg / ml). The effect of paclitaxel at (o) without paclitaxel, (·) 28 µM, (Δ) control (cells alone); n = 3. Figure 3B is a graph showing the concentration dependence over time of inhibition by paclitaxel of superoxide anion production in neutrophils induced by plasma opsonized CPPD crystals; n = 3. Figure 3C is a graph depicting the effect of LY290181 on the generation of superoxide anions in neutrophils induced by CPPD crystals.

Figure 4A is a graph showing the chemiluminescence response of neutrophils (5 x 10⁶ cells / ml) in response to plasma opsonized zymosan (1 mg / ml). Effect of paclitaxel at (O) without paclitaxel, (·) 28 µM; n = 3. Figure 4B is a graph showing superoxide anion production in neutrophils induced by plasma opsonized zymosan. Effect of paclitaxel at (o) without paclitaxel, (·) 28 µM, (Δ) control (cells alone); n = 3.

Figure 5A is a graph showing myeloperoxidase release from neutrophils (5 x 10⁶ cells / ml) in response to plasma opsonized CPPD crystals (50 mg / ml). Effect of paclitaxel at (o) without paclitaxel, (·) 28 µM, (Δ) control (cells alone), (A) control (cells with paclitaxel at 28 µM); n = 3. Figure 5B is a graph showing the concentration dependence of paclitaxel inhibition of myeloperoxidase release from neutrophils in response to plasma opsonized CPPD crystals; n = 3. Figures 5C and 5D are graphs showing that LY290181 decreases the release of both lysozymes and myeloperoxidase in CPPD crystal-induced neutrophils.

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Figure 6 is a graph depicting synoviocyte proliferation at various concentrations of paclitaxel.

Figure 7 is a graph showing the effects of paclitaxel on keratinocytes in vitro.

Figures 8A and 8B show the effect of paclitaxel on astrocyte morphology. Electron microscopy images revealed well-organized thick filamentous processes in astrocytes from transgenic control animals, whereas transgenic animals treated with paclitaxel had morphologically altered astrocytes. Paclitaxel induced astrocyte cell rounding, thinned cell processes, and reduced cytoplasmic filaments relative to untreated animals.

Figure 9 is a graph showing the viability of EOMA cells treated with concentrations of paclitaxel greater than 10^-8 M.

Figure 10 is a bar graph displaying the percentage of apoptolic EOMA cells in a culture treated with increasing concentrations of paclitaxel.

Figures 11A-11E are graphs depicting the effect of various anti-microtubule agents on synoviocytes after a 24 hour period.

Figures 12A-12H are spots showing the effect of various anti-microtubule agents in inhibiting collagenase expression.

Figures 13A-13H are spots showing the effect of various anti-microtubule agents on proteoglycan expression.

Figures 14A and 14B are two photographs of a CAM having a control thermopaste-treated tumor (unloaded). Briefly, in Figure 14A the central white mass is tumor tissue. Note the abundance of blood vessels entering the tumor from the CAM in all directions. The tumor induces the internal growth of the host vascularization through the production of "angiogenic factors". The tumor tissue expands distally along the blood vessels that supply it. Figure 14B is a view from the inner side of the CAM shown at 15A. Briefly, this consideration demonstrates the radial appearance of the blood vessels entering the tumor like the spokes of a wheel. It should be appreciated that the density of the blood vessels is higher in the vicinity of the tumor than in the normal tissue surrounding the CAM. Figures 14C and 14D are two photographs of a CAM having a tumor treated with thermopaste containing 20% ​​paclitaxel. Briefly, in Figure 14C the central white mass is tumor tissue. The paucity of blood vessels in the vicinity of the tumor tissue should be noted. The sustained release of the anti-microtubule agent is able to overcome the angiogenic stimulus produced by the tumor. The tumor itself is poorly vascularized and progressively decreasing in size. Figure 14D is taken from the underside of the CAM shown at 14C and demonstrates the disruption of blood flow in the tumor when compared to a control tumor tissue. It should be appreciated that the density of the blood vessels is reduced in the vicinity of the tumor and is more dispersed than that of the normal surrounding CAM tissue.

Figure 15A is a photograph showing a shellless egg culture on day 6. Figure 15B is a computer-displayed digitized image taken with a stereomicroscope of live unstained capillaries (1040x). Figure 15C is a photograph of a corrosion patch showing chorioalentoic membrane (CAM) microvascularization that is fed by larger underlying vessels (arrows; 1300x). Figure 15D is a photograph showing a 0.5mm thick section of plastic cut transversely through the CAM, and recorded at the light microscope level. This photograph shows the composition of the CAM, including an outer double-layer ectoderm (Ec), a mesoderm (M) containing capillaries (arrow) and scattered adventitious cells, and a single-layer endoderm (En) (400x). Figure 15E is a photograph at the electron microscope level (3500x) showing a typically capillary structure showing thin-walled endothelial cells (arrow heads) and an associated pericyte.

Figures 16A, 16B, 16C and 16D are a series of digitized images of four different unstained CAMs taken after 48 hours of exposure to 10 µg of paclitaxel per 10 ml of methylcellulose. The transparent methylcellulose disk (*) containing paclitaxel is present in each CAM and is placed on a singular avascular zone (A) with surrounding blood islands (Is). These avascular areas extend beyond the disc and are typically about 6mm in diameter. Figure 16D illustrates the typical "kinking" effect (arrow heads) of both small and large vessels being redirected outward from the periphery of the avascular zone.

Figure 17A is a photograph (= 400x) showing capillaries (arrowheads) immediately peripheral to the avascular zone exhibiting numerous mitosis arrested endothelial cells. Ectoderm (Ec); mesoderm (M); endoderm (In). Figure 17B (= 400x) shows that within the avascular zone itself the typical capillary structure has been removed and there are numerous extravasated blood cells. Figure 17C (= 400x) shows that in the central zone of the avascular zone, red blood cells are scattered throughout the mesoderm.

Figure 18A (= 2,200x) shows a small capillarity that is located underlying the ectodermal layer (Ec) that has three endothelial cells arrested in mitosis (*). Various other types of cells both in the

ES 2 207 451 T3 ectoderm and mesoderm are also arrested in mitosis. Figure 18B (= 2,800x) shows that the early avascular phase contains extravasated blood cells underlying the ectoderm; these blood cells are interspersed with presumably endothelial cells (*) and their procedures. Degradative cellular vacuoles (arrowheads). Figure 18C (= 2,800x) shows that in response to paclitaxel, the ectomesodermal interface has become populated with cells in various stages of degradation containing vacuoles and dense granules (arrowheads).

Figure 19A schematically depicts the transcriptional regulation of matrix metalloproteinases. Figure 19B is a blot showing that IL-1 stimulates the transcriptional activity of AP-1. Figure 19C is a graph showing that IL-1 induced decreased binding activity in chondrocyte lysates that were previously treated with paclitaxel.

Figure 20 is a blot showing that IL-1 induction increases levels of collagenase and stromelysin in RNA in chondrocytes, and that this induction can be inhibited by pretreatment with paclitaxel.

Figure 21 is a bar graph depicting the effects of paclitaxel on normal chondrocyte viability in vitro.

Figure 22 is a graph depicting the observed pseudo-first order kinetic degradation of paclitaxel (20 μg / ml^-1) in solutions of 10% IIIpeCD and 10% HPyCd at 37 ° C and pH of 3.7 and 4.9, respectively.

Figure 23 is a graph showing phase solubility of cyclodextrins and paclitaxel in water at 37 ° C.

Figure 24 is a graph showing second order representations of the complexation of paclitaxel and yCD, HpeCD or HPyCD at 37 ° C.

Figure 25 is a table showing the melting temperature, enthalpy, molecular weight, polydispersity, and intrinsic viscosity of a PDLLA-PEG-PDLLA composition.

Figure 26 is a graph displaying PDLLA-PEG-PDLLA and PEG DSC thermograms. The heating rate was 10 ° C / minute. See Figure 30 for melting temperatures and enthalpies.

Fusion 27 is a graph showing the cumulative release of paclitaxel from PDLLAPEG-PDLLA cylinders containing 20% ​​paclitaxel in PBS-albumin buffer at 37 ° C. The error bars represent the standard deviation of 4 samples. PEG 40% cylinders were discontinued after 4 days due to disintegration.

Figures 28A, 28B and 28C are graphs showing the change in dimensions, length (A), diameter (B) and dry weight (C) of PDLLA-PEG-PDLLA cylinders with 20% paclitaxel content during release. in vitro of paclitaxel at 37 ° C.

Figure 29 is a table showing mass loss and polymer composition change of PDLLA-PEG-PDLLA cylinders (containing 20% ​​paclitaxel) during buffer release in PBS-albumin 37 ° C.

Figure 30 is a graph showing gel permeation chromatograms of PDLLA-PEGPDLLA cylinders (20% PEG, 1 mm diameter) with a 20% paclitaxel content during release in PBS-albumin buffer at 37 ° C.

Figures 31A, 31B, 31C and 31D are SEMs of PDLLA-pEG-PDLLA cylinders (containing 20% ​​paclitaxel, 1 mm diameter) before and during the release of paclitaxel. A: 20% PEG, day 0; B: 30% PEG, day 0; C: 20% PEG, day 69; D: 30% PEG, day 69.

Figure 32 is a graph showing the cumulative release of paclitaxel from mixtures of PDLLA: PCL containing 20% ​​paclitaxel and PCL in PBS-albumin buffer at 37 ° C. The error bars represent the standard deviations of 4 samples.

Figure 33 is a graph showing, over time, the release of paclitaxel from PCL pastes in PBS at 37 ° C. PCL pastes contain paclitaxel microparticles and various additives prepared using 140 mesh. Error bars represent the standard deviation of 3 samples.

Figure 34 is a graph depicting paclitaxel release time courses from paclitaxel-gelatin-PCL pastes in PBS at 37 ° C. This graph shows the effects of gelatin concentration (# 140 mesh) and paclitaxel-gelatin microparticle size (1: 1) prepared using a # 140 mesh or # 60 mesh. The error bars represent the 3-sample standard deviation.

Figures 35A and 35B are graphs showing the effect of additives (17A; 140 mesh) and the size of the microparticles (17B; 140 or 60 mesh) and the proportion of the additive (140 mesh) on the swelling behavior of PCL pastes containing 20% ​​paclitaxel after suspension in distilled water at 37 ° C.

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Measurements for paste prepared with 270 µm microparticles in paclitaxel-gelatin and paste containing 30% gelatin were interrupted after 4 hours due to disintegration of the matrix. The error bars represent the standard deviation of 3 samples.

Figures 36A, 36B, 36C and 36D are scanning electron micrographs of paclitaxel-gelatin PCL pastes (20:20:60) before (36A) and after (36B) suspension in distilled water at 37 ° C for 6 hours . Micrographs 36C and 36D are higher magnifications of 36B, showing the intimate association of paclitaxel (in the form of rods) and the gelatin matrix.

Figures 37A and 37B are representative photomicrographs of CAMs treated with gelatin-PCL (37A) and paclitaxel-gelatin-PCL (20:20:60; 37B) pastes showing areas of avascularization in paclitaxel-treated CAM.

Figure 38 is a graph showing phase solubility of cyclodextrins and paclitaxel in water at 37 ° C.

Figure 39 is a graph showing second order representations of the complexation of paclitaxel and yCD, HpeCD or HPyCD at 37 ° C.

Figure 40 is a graph showing phase solubility for paclitaxel at 37 ° C and hydroxypropyl-e-cyclodextrin in solutions in water: ethanol, 50:50.

Figure 41 is a graph showing dissolution rate profiles of paclitaxel in 0, 5, 10, or 20% HPyCD solutions at 37 ° C.

Figure 42 is a graph representing the observed pseudo-first order kinetic degradation of paclitaxel (20 μg / ml) in 10% HP / 1CD and 10% HPyCD solutions at 37 ° C and pH 3.7 and 4.9, respectively.

Figures 43A and 43B, respectively, are two graphs showing the release of paclitaxel from EVA films, and the percentage of paclitaxel that remains in those same films over time. Figure 43C is a graph showing swelling of EVA / F127 films without paclitaxel over time. Figure 43D is a graph showing swelling of EVA / Span 80 films without paclitaxel over time. Figure 43E is a graph showing the strain vs. stress curve for various EVA / F127 blends.

Figure 44 is a graph showing the effect of polymeric microsphere plasma opsonization on the chemiluminescence response of neutrophils (20 mg / ml microspheres in 0.5 ml cells (conc. 5x10⁶cells / ml)) relative to PCL microspheres.

Figure 45 is a graph showing the effect of precoating +/- 2% Pluronic F127 plasma on neutrophil chemiluminescence response (5x10⁶ cells / ml) relative to PCL microspheres.

Figure 46 is a graph showing the effect of precoating +/- 2% Pluronic F127 plasma on the chemiluminescence response of neutrophils (5x10⁶ cells / ml) relative to PMMA microspheres.

Figure 47 is a graph showing the effect of precoating +/- 2% Pluronic F127 plasma on the response to neutrophil chemiluminescence (5x10⁶ cells / ml) relative to PLA microspheres.

Figure 48 is a graph showing the effect of precoating +/- 2% Pluronic F127 plasma on the chemiluminescence response of neutrophils (5x10⁶ cells / ml) relative to EVA: PLA microspheres.

Figure 49 is a graph showing the effect of precoating IgG (2 mg / ml) or 2% Pluronic F127 and then IgG (2 mg / ml) on the chemiluminescence response of neutrophils to PCL microspheres. .

Figure 50 is a graph showing the effect of precoating IgG (2 mg / ml) or 2% Pluronic F127 and then IgG (2 mg / ml) on the chemiluminescence response of neutrophils relative to PMMA microspheres. .

Figure 51 is a graph showing the effect of precoating IgG (2 mg / ml) or 2% Pluronic F127 and then IgG (2 mg / ml) on the chemiluminescence response of neutrophils to PVA microspheres. .

Figure 52 is a graph showing the effect of precoating IgG (2 mg / ml) or 2% Pluronic F127 and then IgG (2 mg / ml) on the chemiluminescence response of neutrophils relative to EVA microspheres. : PLA.

Figure 53A is a graph showing release rate profiles from polycaprolactone microspheres containing 1%, 2%, 5% or 10% paclitaxel in phosphate buffered saline at 37 ° C. The figure

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53B is a photograph showing a CAM treated with control microspheres. Figure 53C is a photograph showing a CAM treated with microspheres containing 5% paclitaxel.

Figure 54 is a graph showing the range of particle sizes for control microspheres (PLLA: GA

- 85:15).

Figure 55 is a graph showing the particle size range for microspheres containing 20% ​​paclitaxel (PLLA: GA-85:15).

Figure 56 is a graph showing the range of particle sizes for control microspheres (PLLA: GA

- 85:15).

Figure 57 is a graph showing the particle size range for microspheres containing 20% ​​paclitaxel (PLLA: GA-85:15).

Figures 58A, 58B and 58C are graphs showing paclitaxel release rate profiles from varying ranges of microsphere sizes and various PLLA and GA ratios.

Figures 59A and 59B are graphs showing the release rate profiles of paclitaxel from microspheres with various ratios of PLLA and GA.

Figures 60A and 60B are graphs showing the release rate profiles of paclitaxel from microspheres with various ratios of PLLA and GA.

Figures 61A, 61B and 61C are graphs showing the release rate profiles of paclitaxel from microspheres of varying size and various ratios of PLLA and GA.

Figure 62 is a graph depicting the release of paclitaxel from paclitaxel-nylon microcapsules.

Figures 63A and 63B are photographs of fibronectin-coated PLLA microspheres on bladder tissue (63A) and poly (L-lysine) microspheres on bladder tissue.

Figure 75 is a graph depicting the effect of high dose interval paclitaxel therapy on the progression of clinical symptoms in transgenic mice. The transgenic mice were treated with 20 mg / kg of paclitaxel once a week for 4 weeks (weeks 0, 1, 2 and 3) and were monitored for 10 weeks, every other day, with titrations determined for each symptom. Data represent mean score (cumulative for all symptoms) for paclitaxel-treated transgenic mice (n = 5) and control mice (n = 3). Treatment with paclitaxel reduced the deterioration caused by the over-expression of DM20 in the transgenics, while the control mice deteriorated very rapidly, so that 2 out of 3 mice did not survive at the end of the experimental protocol (as indicated).

Figures 76A and 76B show a paclitaxel paste applied perivascularly (in the adventitia of the blood vessel) in the rat carotid artery model. The adventitious surface of the left common carotid artery was treated with 2.5 mg of a control paste (76A) or a paste containing 20% ​​paclitaxel (76B). Control arteries showed increased arterial wall thickness due to hyperproliferation of smooth muscle cells, whereas the paclitaxel-containing paste-treated artery did not show any evidence of intimal thickening.

Figures 77A and 77B depict the proximity effect of perivascular paclitaxel paste in the rat carotid artery model. The paclitaxel-containing paste applied immediately adjacent to the perivascular area of ​​the vessel prevented restenosis; however, when the paste was not directly adjacent to the vessel wall, neointimal hyperplasia of the vessel wall was evident.

Figures 78A, 78B and 78C show the effect of paclitaxel on GFAP staining of astrocytes. The brain sections of normal animals and transgenic animals (which developed a neurological disease similar to multiple sclerosis) treated with vehicle or paclitaxel were stained with GFAP (a marker for activated astrocytes) and were examined histologically. In the control transgenic mice there was an increase in the number of astrocytes and the levels of total GFAP compared to the sections of the normal brains. However, the morphology of the cells was similar. Brain sections from transgenic mice treated with paclitaxel showed decreased astrocyte numbers and GFAP levels compared to untreated transgenic animals. Histologically, there is cellular rounding and thinning of the stellate procedures in astrocytes.

Figures 79A and 79B are graphs showing that paclitaxel inhibits T cell stimulation in response to the basic protein peptide myelin (GP68-88) and ConA. A 48 hour culture of RT1 T cell proliferation was performed with GP68-88 (A) or ConA (B) as stimulagens. Paclitaxel and its vehicle (micelles) were added at graduated concentrations at the start of antigen challenge or 24 hours later. Paclitaxel inhibited T cell proliferation at concentrations as low as 0.02 µM, independent of stimulagen.

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Figures 80A, 80B, 80C and 80D are graphs showing that tubercidin and paclitaxel inhibit IL-1 and TNF-induced NF-κΒ activity.

Figures 81A and 81B are graphs showing the effect of increasing concentrations of paclitaxel or campothecin on cell growth of human prostate cancer cells (LNCaP) (2x103 cells / well) measured by crystal violet staining (0.5%). and quantification by absorbance at 492 nm. The growth percentage is expressed as% relative to controls and an average of 8 results is provided.

Detailed description of the invention

Before stating the invention, it may be helpful for your understanding to set out the definitions of certain terms that will be used later.

"Inflammatory disease", as used herein, refers to any of a number of diseases that are characterized by vascular changes: edema and neutrophil infiltration (eg, acute inflammatory reactions), tissue infiltration by cells mononuclear; destruction of tissues by inflammatory cells, connective tissue cells and their cellular products; and attempts at repair by replacement of connective tissues (eg, chronic inflammatory reactions). Representative examples of these diseases include many common medical conditions such as arthritis, atherosclerosis, psoriasis, inflammatory bowel disease, multiple sclerosis, surgical adhesions, restenosis, tuberculosis, graft rejection, and chronic inflammatory respiratory diseases (eg, asthma, pneumoconiosis, disease chronic obstruction lung, nasal polyps and pulmonary fibrosis).

"Anti-microtubule agents" should be understood to include any protein, peptide, chemical, or other molecule that impairs the function of microtubules, for example, through the prevention or stabilization of polymerization. A wide variety of methods can be used to determine the anti-microtubule activity of a particular compound including, for example, the assays described by Smith et al. (Cancer Lett 79 (2): 213-219, 1994) and Mooberry et al., (Cancer Lett. 96 (2): 261-266, 1995).

As indicated above, the use according to the invention is useful for treating or preventing inflammatory diseases, comprising the step of delivering an anti-microtubule agent to the site of inflammation. Briefly, a wide variety of agents can be delivered to a site of inflammation (or potential site of inflammation) with or without a vehicle (eg, a polymer or ointment) in order to treat or prevent an inflammatory disease. Representative examples of such agents include taxanes (eg, paclitaxel (discussed in more detail below) and docetaxel) (Schiff et al., Nature 277: 665-667, 1979; Long and Fairchild, Cancer Research 54: 4355-4361, 1994; Ringel and Horwitz, J. Natl. Cancer Inst. 83 (4): 288-291, 1991; Pazdur et al., Cancer Treat. Rev. 19 (4): 351-386, 1993), campothecin, electerobine ( for example, US Pat. No. 5,473,057, sarcodichthyins (including sarcodichthyin A), epothilones A and B (Bollag et al., Cancer Research 55: 2325-2333, 1995), discodermolide (ter Haar et al., Biochemistry 35: 243-250, 1996 ), deuterium oxide (D2O) (James and Lefebvre, Genetics (130 (2): 305-314, 1992; Sollott et al., J. Clin, Invest. 95: 1869-1876, 1995), hexylene glycol (2- methyl-2,4-pentanediol (Oka et al., Cell Struct. Funct. 16 (2): 125134, 1991), tubercidin (7-deazaadenosine) (Mooberry et al., Cancer lett. 96 (2): 261-266, 1995), LY290181 (2-amino-4- (3-pyridyl) -4H-naphtho (1,2-b) pyran-3-carbonitrile) panda et al., J. Biol Chem. 272 ​​(12): 7681-7687, 1997; Wood et al., Mol. Pharmacol. 52 (3): 437-444, 1997), aluminum fluoride (Song et al., J. Cell. Sci. Suppl. 14: 147-150, 1991), ethylene glycol bis- (succinimidylsuccinate) (Caplow and Shanks, J. Biol. Chem. 265 (15): 8935-8941, 1990), glycine ethyl ester (Mejillano et al., Biochemistry 31 (13): 3478-3483, 1992), monoclonal anti-idiotypic antibodies (Leu et al., Proc. Natl. Acad. Sci. USA 91 (22): 10690-10694, 1994), microtubule array favoring protein (taxol-like protein, TALP) (Hwang et al., Biochem Biophys. Res. Commun. 208 (3): 1174-1880, 1995), cell swelling induced by hypotonic conditions (190 mosmol / l), insulin (100 nmol / l) or glutamine (10 mmol / l) (Haussinger et al., Biochem Cell. Biol . 72 (1-2): 12-19, 1994), dynein binding (Ohba et al., Biochim. Biophys. Acta 1158 (3): 323-332, 1993), gibberellin (Mita and Shibaoka, Protoplasma 119 ( 1/2): 100-109, 1984), XCHO1 (kinesin-like protein) (Yonetani et al., Mol. Biol. Cell 7 (suppl): 211A, 1996), lysophosphatidic acid (Cook et al., Mol. Biol. Cell 6 (suppl): 260A, 1995), lithium ion (Bhattacharyya and Wolff, Biochem. Biophys. Res. Commun. 73 (2): 383-390, 1976) components of plant cell walls (eg, poly-1-lysine and extensin) (Akashi et al., Plant 182 (3): 363-369, 1990), glycerol buffers (Schilstra et al., Biochem. J. 277 (Pt. 3): 839,847, 1991 ; Farrell and Keates, Biochem. Cell. Biol. 68 (11): 1256-1261; 1990); Lopes et al., J. Cell. Biochem. 43 (3): 281-291, 1990), Triton X-100 microtubule stabilizing buffer (Brown et al., J. Cell Sci. 104 (Pt. 2): 339-352, 1993; Safiejko-Mroczka and Bell, J. Histochem. Cytochem. 44 (6): 641-656, 1996), microtubule-associated proteins (eg, MAP2, MAP4, tau, big tau, ensconsin, elongation factor 1-alpha (EF-1a) and E -MAP-115) (Burgess et al., Cell Motil. Cytoskeleton 20 (4): 289-300, 1991; Saoudi et al., J. Cell. Sci. 108 (Pt. 1): 357-367, 1995; Bulinski and Bossler, J. Cell. Sci. 107 (Pt. 10): 2839-2849, 1994; Ookata et al., J. Cell Ciol 128 (5): 849-862, 1995; Boyne et al., J. Comp. Neurol. 358 (2): 279-293, 1995; Ferreira and Caceres, J. Neurosci. 11 (2): 392-400, 1991; Thurston et al., Chromosoma 105 (1): 20-30, 1996; Wang et al., Brain Res. MOl. Brain Res. 38 (2): 200-208, 1996; Moore and Cyr, Mol. Biol. Cell 7 (suppl): 221-A, 1996; Masson and Kreis, J. Cell Biol. 123 (2), 357-371, 1993), cellular entities (eg, histone H1, myelin basic protein, and kinetochore) (Saoudi et al., J. Cell. Sci. 108 (pt. 1): 357-367, 1995; Simerly et al., J. Cell Biol. 111 (4): 1491-1504, 1990), endogenous microtubule structures (eg, axonemal structures, plugs and GTP caps) (Dye et al., Cell Motil. Cytoskeleton 21 (3): 171-186, 1992; Azhar and Murphy, Cell Motil Cytoskeleton 15 (3): 156-161, 1990; Walker et al., J. Cell Biol. 114 (1): 73-81, 1991; Drechsel and Kirschner, Curr. Biol. 4 (12): 1053-1061, 1994), tubule-stable only polypeptides (eg, STOP145 and STOP220) (Pirollet et al., Biochim. Biophys. Acta 1160 (1):

ES 2 207 451 T3

113-119, 1992; Pirollet et al., Biochemistry 31 (37): 8849-8855, 1992; Bose et al., Proc. Natl. Acad. Sci. USA 93 (5): 2125-2130, 1996; Margolis et al., EMBO J. 9 (12): 4095-4102, 1990) and mitotic force tension (Nicklas and Ward, J. Cell Biol. 126 (5): 1241-1253, 1994), as well as any analogs and derived from any of the foregoing. Such compounds can act by depolymerizing microtubules (eg, colchicine and vinblastine) or by stabilizing microtubule formation (eg, paclitaxel).

Within a preferred embodiment of the invention, the therapeutic agent is paclitaxel, a compound that disrupts microtubule formation by binding to tubulin to form abnormal mitotic needles. Briefly, paclitaxel is a highly derived diterpenoid (Wani et al., J. Am. Chem. Soc. 93: 2325, 1971) which has been obtained from the cultivated and dried bark of Taxus brevifolia (Pacific yew) and Taxomyces Andreanae and Endophytic Fungus of the Pacific yew (Stierle et al., Science 60: 214-216, 1993 ). "Paclitaxel" (to be understood herein to include prodrugs, analogs and derivatives such as, for example, TAXOL^®, TAXOTERE^®, Docetaxel, 10-deacetyl analogs of paclitaxel and 3'-N-desbenzoyl-3'-Nt-butoxycarbonyl analogs of paclitaxel) can be readily prepared using techniques known to those skilled in the art (see, for example, Schiff et al. al., Nature 277: 665-667, 1979; Long and Fairchild, Cancer Research 54: 4355-4361, 1994; Ringel and Horwitz, J. Natl. Cancer Inst. 83: (4): 288-291, 1991; Pazdur et al., Cancer Treat, Rev. 19 (4): 351-386, 1993; 94/07882, WO 94/07881, WO 94/07880, WO 94/07876; WO 93/23555, WO 93/10076, WO 94/00156, WO 93/24476, EP 590267, WO 94/20089; US patents Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, 5,254,580, 5,412,092, 5,395,850, 5,380,751, 5,350,866, 4,857,653, 5,272,171, 5,411,984, 5,248,796, 5,422,364, 5,300,638, 5,294,637, 5,362,831, 5,440,056, 4,814,470, 5,278,324, 5,352,805, 5,411. 984, 5,059,699, 4,942,184; Tetrahedron Letters 35 (52): 9709-9712, 1994; J. Med. Chem. 35: 4230-4237, 1992; J. Med. Chem. 34: 992-998, 1991; J. Natural Prod. 57 (10): 1404-1410, 1994; J. Natural Prod. 57 (11): 1580-1583, 1994; J. Am. Chem. Soc. 110: 6558-6560, 1988), or can be obtained from a variety of commercial sources including, for example, Sigma Chemical Co., St. Louis, Missouri (T7402 - from Taxus brevifolia).

Representative examples of such paclitaxel derivatives or analogs include 7-deoxy-docetaxol, 7,8-cyclopropataxanes, N-substituted 2-azetidones, 6,7-epoxypaclitaxels, 6,7,10-deacetoxitaxol modified paclitaxels, 10-deacetyltaxol (from of 10-deacetyl-baccatin III), phosphonooxy and carbonate derivatives of taxol, 2,7-di (1,2-sodium benzenedicarboxylate) of taxol, derivatives of 10-deacetoxy-11,12-dihydrotaxol-10,12 ( 18) -diene, 10-deacetoxitaxol, Protaxol (2'- and / or 7-O-ester derivatives, 2'- and / or 7-O-carbonate derivatives), asymmetric synthesis of taxol side chains, fluorotaxols, 9-deoxotaxane, 13-acetyl- 9-deoxo-baccatin III, 9-deoxotaxol, 7-deoxy-9-deoxotaxol, 10-deacetoxy-7-deoxy-9-deoxotaxol, derivatives containing hydrogen or one acetyl group and one hydroxy and tert-butoxycarbonylamino, derivatives of 2'- sulfonated acryloyltaxol and sulfonated 2'O-acyl-acid-taxol, succinyltaxol, 2'-Y-aminobutyryltaxol formate, 2'-acetyl-taxol, 7-acetyl-taxol, 7-glycine-carbamate-taxol, 2'-OH-7-PEG (5000) -carbamatotaxol, 2'-benzoyl and 2'-7-dibenzoyl taxol derivatives , other prodrugs (2'-acetyltaxol; 2 ', 7-diacetyltaxol; 2'succinyltaxol; 2' - (beta-alanyl) -taxol); 2'-gamma-aminobutyryltaxol formate; ethylene glycol derivatives of 2'-succinyltaxol; 2'-glutaryltaxol; 2 '- (N, N-dimethylglycyl) taxol; 2 '- (2- (N, N-dimethylamino) propionyl) taxol; 2'-orthocarboxybenzoyl-taxol; 2'-aliphatic-carboxylic acid derivatives of taxol, prodrugs {2 '- (N, N- -dimethylaminopropionyl) taxol, 2' - (N, N-dimethylglycyl) taxol, 7- (N, N-dimethylglycyl) taxol, 2 ', 7-di- (N, N-dimethylglycyl) -taxol, 7- (N, N-dimethylaminopropionyl) taxol, 2', 7-di (N, N-diethylaminopropionyl) taxol, 2 '- (L-glycyl ) taxol, 7- (L-glycyl) taxol, 2 ', 7-di (L-glycyl) taxol, 2' - (L-alanyl) taxol, 7- (L-alanyl) taxol, 2 ', 7 -di (L-alanyl) taxol, 2 '- (L-leucyl) -taxol, 7- (L-leucyl) taxol, 2', 7-di (L-leucyl) taxol, 2 '- (Lisoleucyl) taxol, 7- (L-isoleucyl) taxol, 2 ', 7-di (L-isoleucyl) taxol, 2' - (L-valyl) taxol, 7- (L-valyl) taxol, 2 ', 7-di (L- valyl) taxol, 2 '- (L-phenylalanyl) taxol, 7- (L-phenylalanyl) taxol, 2', 7-di (L-phenylalanyl) taxol, 2 '- (L-prolyl) taxol, 7- (L -prolyl) taxol, 2 ', 7-di (L-prolyl) taxol, 2' - (L-lysyl) taxol, 7- (L-lysyl) taxol, 2 ', 7-di (L-lysyl) taxol, 2 '- (L-glutamyl) taxol, 7- (L-glutamyl) taxol, 2', 7di (L-glutamyl) taxol, 2 '- (L-arginyl) taxol, 7- (L-arginyl) taxol, 2 ', 7-di (L-arginyl) taxol}, Taxol analogs with modified phenylisoserine side chains, taxotere (N-desbenzoyl-N-tert- (butoxycarbonyl) -10-deacetyltaxol, and taxanes (eg, baccatin III, cephalomin, 10-deacetylbaccatin III, brevifoliol, yunantaxusin, and taxusin).

Representative examples of microtubule depolymerization (or destabilization or disruption) agents include Nocodazol (Ding et al., J. Exp. Med. 171 (3): 715-727, 1990; Dotti et al., J. Cell Sci. Suppl. 15: 75-84, 1991; Oka et al., Cell Struct. Funct. 16 (2): 125-134, 1991; Wiemer et al., J. Cell. Biol. 136 (1): 71-80, 1997); Quitocalasin B (Illinger et al., Biol. Cell 73 (2-3): 131-138, 1991); Vinblastine (Ding et al., J. Exp. Med. 171 (3): 715-727. 1990: Dirk et al., Neurochem. Res. 15 (11): 1135-1139, 1990; Illinger et al., Biol Cell 73 (2-3): 131-138, 1991; Wiemer et al., J. Cell. Biol. 136 (1): 71-80, 1997); Vincristine (Dirk et al., Neurochem Res. 15 (11): 1135-1139, 1990; Ding et al., J. Exp. Med. 171 (3): 715-727, 1990); Colchicine (Allen et al., Am. J. Physiol, 261 (4 Pt. 1): L315-L321, 1991; Ding et al., J. Exp. Med. 171 (3): 715-727, 1990; Gonzalez et al., Exp. Cell. Res. 192 (1): 10-15, 1991; Stargell et al., Mol. Cell. Biol. 12 (4): 14431450, 1992); CI 980 (Colchicine analog) (Garcia et al., Anticancer Drugs 6 (4): 533-544, 1995); Colcemid (Barlow et al., Cell. Motil. Cytoskeleton 19 (1): 9-17, 1991; Meschini et al., J. microsc. 176 (Pt. 3): 204-210, 1994; Oka et al., Cell Struct. Funct. 16 (2): 125-134, 1991); Podophyllotoxin (Ding et al., J. Exp. Med. 171 (3): 715-727, 1990); Benomyl (Hardwick et al., J. Cell. Biol. 131 (3): 709-720, 1995; Shero et al., Genes Dev. 5 (4): 549-560, 1991); Orizalina (Stargell et al., Mol. Cell. Biol. 12 (4): 1443-1450, 1992); Majusculamide C (Moore, J. Ind. Microbiol. 16 (2): 134-143-1996); Demecolcin (Van Dolah and Ramsdell, J. Cell. Physiol. 166 (1): 49-56, 1996; Wiemer et al., J. Cell. Biol. 136 (1): 71-80, 1997); and methyl-2-benzimidazolecarbamate (MBC) (Brown et al., J. Cell. Biol. 123 (2): 387-403, 1993).

Formulations

As indicated above, the anti-microtubule therapeutics described herein can be formulated in a variety of ways and thus may additionally comprise a

ES 2 207 451 T3 vehicle. In this regard, a wide variety of carriers of polymeric or non-polymeric origin can be selected.

For example, within one embodiment of the invention a wide variety of polymeric carriers can be used to contain and / or deliver one or more of the above-discussed therapeutic agents including, for example, both biodegradable and non-biodegradable compositions. Representative examples of biodegradable compositions include albumin, collagen, gelatin, hyaluronic acid, starch, cellulose (methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate-phthalate, cellulose acetate-succinate, hydroxypropylcellulose, polysaccharide, cellulosexamyl acetate, hydroxypropyl cellulose). fibrinogen, poly (D, L-lactide), poly (D, L-lactide-Co-glycolide), poly (glycolide), poly (hydroxybutyrate, poly (alkylcarbonate) and poly (orthoesters), polyesters, poly (hydroxyvaleric acid), polydioxanone, poly (ethylene terephthalate), poly (malic acid), poly (tartronic acid), polyanhydrides, polyphosphazenes, poly (amino acids) and their copolymers (see generally Illum, L. Davids, SS (eds.) "Polymers in Controlled Drug Delivery" Wright, Bristol, 1987; Arshady, J. Controlled Release 17: 1-22, 1991; Pitt, Int. J. Phar. 59: 173-196, 1990; Holland et al., J. Controlled Release 4: 155-0180, 1986). Representative examples of non-degradable polymers include poly (ethylene-vinyl acetate) copolymers ("EVA"), silicone rubber, acrylic polymers (poly (acrylic acid), poly (methacrylic acid), poly (methyl methacrylate), poly (alkyl cyanoacrylate)), polyethylene, polypropylene, polyamides (nylon 6,6), polyurethane, poly (ester-urethanes), poly (ether-urethanes), poly (ester-urea), polyethers, poly (ethylene oxide) , poly (propylene oxide), Pluronics and poly (tetramethylene glycol)), silicone rubbers and vinyl polymers (polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate phthalate). Polymers that are anionic can also be developed (e.g. alginate, carrageenan, carboxymethyl cellulose and poly (acrylic acid)) , or cationic (eg, chitosan, poly-L-lysine-polyethyleneimine, and poly (allylamine)) (see generally Dunn et al., J. Applied Polymer Sci. 50: 353,365, 1993; Cascone et al., J. Materials Sci .: Materials in Medicine 5: 770-774, 1994; Shiraishi et al., Biol. Pharm. Bull. 16 (11): 1164-1168, 1993; Thacharodi and Rao, Int'l J. Pharm. 120: 115,118, 1995; Miyazaki et al., Int'l J. Pharm. 118: 257-263, 1995). Particularly preferred polymeric carriers include poly (ethylene-vinyl acetate) oligomers and polymers, poly (D, L-lactic acid), poly (L-lactic acid) oligomers and polymers, poly (glycolic acid), acid copolymers lactic and glycolic acid, poly (caprolactone), poly (valerolactone), polyanhydrides, copolymers of poly (caprolactone) or poly (lactic acid) with polyethylene glycol (eg MePEG) and their mixtures.

Polymeric carriers can be patterned in a variety of ways, with desired release characteristics and / or with desired specific properties. For example, polymeric carriers can be modeled to release a therapeutic agent upon exposure to a specific trigger event such as pH (see eg, Heller et al., "Chemically Self-Regulated Drug Delivery Systems," in Polymers in Medicine III, Elsevier Science Publishers BV, Amsterdam, 1988, pp. 175-188; Kang et al., J. Applied Polymer Sci. 48: 343-354, 1993; Dong et al., J. Controlled Release 19: 171-178 , 1992; Dong and Hoffman, J. Controlled Release 15: 141-152, 1991; Kim et al., J. Controlled Release 28: 143-152, 1994; Cornejo-Bravo et al., J. Controlled Release 33: 223-229, 1995; Wu and Lee, Pharm. Res. 10 (10): 1544-1547, 1993; Serres et al., Pharm. Res. 13 (2): 196-201, 1996; Peppas, "Fundamentals of pH- and Temperature-Sensitive Delivery Systems", in Gurny et al. (eds), Pulsatile Drug Delivery, Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart, 1993, p. 41-55; Doelker, "Cellulose Derivatives", 1993, in Peppas and Langer (eds), Biopolymers I, Springer-Verlag, Berlin). Representative examples of pH-sensitive polymers include polyacrylic acid and derivatives thereof (including, for example, homopolymers such as polyaminocarboxylic acid); polyacrylic acid; polyacrylic acid; copolymers of such homopolymers, and copolymers of polyacrylic acid and acrylic monomers, as previously discussed. Other pH sensitive polymers include polysaccharides such as cellulose acetate phthalate; Hydroxypropylmethylcellulose Phthalate; Hydroxypropylmethylcellulose acetate-succinate; cellulose acetate-trimethylate and chitosan. Still other pH sensitive polymers include any mixture of a pH sensitive polymer and a water soluble polymer.

Similarly, polymeric carriers can be tempered to be temperature sensitive (see, for example, Chen et al. Publication, "Novel Hydrogels of a Temperature-Sensitive Pluronic Grafted of a Bioadhesive PolyacrylidAcidBackbone for Vaginal Drug Delivery", in Proceed, Intern Symp Control Rel Rel Bioact Mater 22: 1 67168, Controlled Release Society, Incl, 1995; Okano, "Molecular Design of Stimuli-Responsive Hydrogels for Temperal Controlled Drug Delivery", in Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 22: 111-112, Controlled Release Society Inc., 1995; Johnston et al., Pharm. Res. 9 (3): 425-433, 1992, Tung, Int'l J. Pharm. 107: 85-90, 1994; Harsh and Gehrke, Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 17: 175-186, 1991; Bae et al., Pharm. Res. 8/4): 531-537, 199; Dinarvand and D'Emanuele, J. Controlled Release 36: 221-227, 1995; Yu and Grainger, “Novel Thermo-sensitive Amphiphilic Gels: Poly N-isopropylacrylamide-co-sodium acrylate-co-nN-alkylacrylamide Network Synthesis and Pjysicochemical Characterization”, Dept. of Chemical & Biological Sci., Oregon Graduate Institute of Science & Technology, Beaverton , OR, p. 820-821; Zhou and Smid, "Physical Hydrogels of Associative Star Polymers", Polymer Research Indstitute, Dept. of Chemistry, College of Environmental Science and Forestry, State Univ. of New York, Syracuse, NY, p. 822-823; Hoffman et al., "Characterizing Pore Sizes and Water" Structure "in Stimuli-Responsive Hydrogels", Center for Bioengineering, Univ. Of Washington, Seattle, WA, p. 828; Yu and Grainger, "Thermosensitive Swelling Behavior in Crosslinked N-isopropylacrylamide Networks: Cationic, Anionic and Ampholytic Hydrogels", Dept. of Chemical & Biological Sci., Oregon Graduate Institute of Science & Technology, Beaverton, OR, p. 829-830; Kim et al., Pharm. Res. 93 (3): 283-290, 1992; Bae et al., Pharm. Res. 8 (5): 624-628, 1991; Kono et al., J. Controlled Release 30: 69-75, 1994; Yoshida et al., J. Controlled Release 32: 97-102, 1994 Okano et al., J. Controlled Release 36: 125-133, 1995; Chun and Kim, J. Controlled Release 38: 39-47, 1996; D'Emanuele and Dinarvand, Int'l J. Pharm. 118: 237-242, 1995; Katono et al., J. Controlled Release 16: 215-228, 1991; Hoffman, "Thermally Reversible Hydrogels Containing Biologically Active Species", in Migliaresi et al. (eds), Polymers in Medicine III, Elsevier Science Publishers BV, Amsterdam, 1988, p. 161-167; Hoffman, "Applications of Thermally Reversible Polymers

ES 2 207 451 T3 and Hydrogels in Therapeutics and Diagnostics ”, in Third International Symposium on Recent Advances in Drug Delivery Sustems, Salt Lake City, UT, Feb. 24-27, 1987, p. 297-305; Gutowska et al., J. Controlled Release 22: 95-104, 1992; Palasis and Gehrke, J. Controlled Release18: 1-12, 1992; Paavola et al., Pharm. Res. 12 (12): 1997-2002, 1995).

Representative examples of thermogelling polymers, and their gelatin temperature (LCST (° C)) include homopolymers such as poly (N-methyl-Nn-propylacrylamide), 19.8; poly (Nn-propylacrylamide), 21.5; poly (N-methylN-isopropylacrylamide, 22.3; poly (Nn-propylmethacrylamide), 28.0; poly (N-isopropylacrylamide), 30.9; poly (Nn-diethylacrylamide), 32.0; poly (N-isopropylmethacrylamide) ), 44.0, poly (N-cyclopropylacrylamide), 45.5, poly (N-ethylmethylacrylamide), 50.0; poly (N-methyl-N-ethylacrylamide), 56.0; poly (N-cyclopropylmethacrylamide), 59.0; poly (N-ethylacrylamide), 72.0. In addition, thermogelling polymers can be made by preparing copolymers between monomers of the foregoing, or by combining such homopolymers with other water-soluble polymers such as acrylic monomers (for example, acrylic acid and its derivatives such as methacrylic acid, acrylate and its derivatives). derivatives such as methacrylate, acrylamide and Nn-butylacrylamide).

Other representative examples of thermogelling polymers include derivatives of cellulose ethers such as hydroxypropyl cellulose, 41 ° C; methyl cellulose, 55 ° C; hydroxypropylmethyl cellulose, 66 ° C and ethylhydroxyethyl cellulose, and Pluronics such as F-127, 10-15 ° C; L-122, 19 ° C, L-92.26 ° C; L-81, 20 ° C and L-61, 24 ° C.

A wide variety of shapes can be modeled by the polymeric carriers of the present invention including, for example, devices in the form of rods, granules, tablets or capsules (see, for example, Goodell et al., J. Hosp. Pharm. 43: 1454-1461, 1986, Langer et al., "Controlled release of macromolecules from Polymers", in Biomedical Polymers, Polymeric Materials and Pharmaceuticals for Biomedical Use, Goldberg, EP, Nakagim, A. (eds) Academic Press, p. 113-137, 1980; Rhine et al., J. Pharm. Sci. 69: 265-270, 1980; Brown et al., J. Pharm. Sci. 72: 1181-1185, 1983; and Bawa et al., J. Controlled Release 1: 259-267, 1985). Therapeutic agents can be occlusively bound in polymer matrices, covalently bound, or encapsulated in microcapsules. Within certain preferred embodiments of the invention, therapeutic compositions are provided in non-capsular formulations such as microspheres (ranging in size in the 1 nanometer to micron range), pastes, various sized filaments, films, and sprays.

Preferably, the therapeutic compositions described herein are modeled in a manner appropriate to the intended use. Within certain aspects of the present invention, the therapeutic composition must be biocompatible and release one or more therapeutic agents over a period of several days to months. For example, "rapid release" or "flash" therapeutic compositions are provided that release more than 10%, 20%, or 25% (w / v) of a therapeutic agent (eg, paclitaxel) over a period of 7 to 10 days. Such "rapid release" compositions must be capable, in certain embodiments, of delivering chemotherapeutic levels (where applicable) of a desired agent. Within other embodiments, "slow release" therapeutic compositions are provided that release less than 1% (w / v) of a therapeutic agent over a period of 7 to 10 days. Additionally, the therapeutic compositions of the present invention should preferably be stable for several months and capable of being produced and maintained under sterile conditions.

In certain aspects of the present invention, therapeutic compositions can be shaped in any size ranging from 50 nm to 500 µη, depending on the particular use. Alternatively, such compositions can also be easily applied in the form of a "spray" that solidifies into a film or coating. Such sprays can be prepared from microspheres of a wide range of sizes, including, for example, 0.1 µm to 3 µm, 10 µm to 30 µm, and 30 µm to 100 µm.

The therapeutic compositions described herein can also be prepared in a variety of "paste" or gel forms. For example, in one embodiment of the invention, therapeutic compositions are provided that are liquid at a temperature (for example, a temperature greater than 37 ° C, such as 40 ° C, 45 ° C, 50 ° C, 55 ° C or 60 ° C) and solid or semi-solid at another temperature (for example, ambient body temperature or any temperature below 37 ° C). Such "thermopastes" can be easily prepared given the description provided herein.

Within still other aspects of the invention, the therapeutic compositions can be formed as a film. Preferably, such films are generally less than 5, 4, 3, 2 or 1 mm thick, more preferably less than 0.75 mm or 0.5 mm thick, and most preferably less than 500 μm to 100 μm in thickness. thickness. Such films are preferably flexible with good tensile strength (eg greater than 50, preferably greater than 100, and more preferably greater than 150 or 200 N / cm²), good adhesive properties (i.e., adheres easily to damp or wet surfaces), and has controlled permeability.

Within additional aspects of the invention, therapeutic compositions can be formulated for topical application. Representative examples include: ethanol; ethanol-glycols mixtures (eg, ethylene glycol or propylene glycol); mixtures of ethanol and isopropyl myristate or ethanol, isopropyl myristate and water (eg, 55: 5:40), mixtures of ethanol and eineol or D-limonene (with or without water); glycols (for example, ethylene glycol or propylene glycol) and glycol mixtures such as propylene glycol and water, phosphatidyl glycerol, dioleylphosphatidyl glycerol, Transcutol^®or terpinolene; mixtures of isopropyl myristate and 1-hexyl-2-pyrrolidone, N-dodecyl-2-piperidinone or 1-hexyl-2-pyrrolidone. Other excipients may also be added, including, for example, acids such as oleic acid and linoleic acid and soaps such as sodium lauryl sulfate. For a more detailed description of what

ES 2 207 451 T3 above see, generally, Hoelgaard et al., J. Contr. Rel. 2: 111, 1985; Liu et al., Pharm. Res. 8: 938, 1991; Roy et al., J. Pharm. Sci. 83: 126, 1992; Ogiso et al., J. Pharm. Sci. 84: 482, 1995; Sasaki et al., J. Pharm. Sci. 80: 533, 1991; Okabe et al., J. Contr. Rel. 32243, 1994; Yokomizo et al., J. Contr. Rel. 38: 267, 1996 Yokomizo et al., J. Contr. Rel. 42:37, 1996; Mond et al., J. Contr. Rel. 33:72, 1994; Michniak et al., J. Contr. Rel. 32: 147, 1994; Sasaki et al., J. Pharm. Sci. 80: 533, 1991; Baker & Hadgraft, Pharm. Res. 12: 993, 1995; Jasti et al., AAPS Proceedings, 1996; Lee et al., AAPS Proceduings, 1996; Ritschel et al., Skin Pharmacol, 4: 235, 1991; and McDaid & Deasy, Int. J. Pharm .. 133: 71, 1996.

Within certain embodiments of the invention, therapeutic compositions may also comprise additional ingredients such as surfactants (eg, Pluronics such as F-127, L-122, L-92, L-81, and L-61).

Within additional aspects of the present invention, polymeric carriers are provided that are adapted to contain and release a hydrophobic compound, such that the carrier contains the hydrophobic compound in combination with a carbohydrate, protein, or polypeptide. Within certain embodiments, the polymeric carrier contains or comprises zones, pockets, or granules of one or more hydrophobic compounds. For example, within one embodiment of the invention, the hydrophobic compounds can be incorporated into a matrix containing the hydrophobic compound, followed by incorporation of the matrix into the polymeric carrier. A variety of matrices can be used in this regard including, for example, carbohydrates and polysaccharides such as starch, cellulose, dextran, methylcellulose, and hyaluronic acid, proteins or polypeptides such as albumin, collagen, and gelatin. Within alternative embodiments, the hydrophobic compounds may be contained in a hydrophobic core and this core contained in a hydrophilic shell.

Other vehicles that can be similarly used to contain and deliver the therapeutic agents described herein include: hydroxypropyl-e-cyclodextrin (Cserhati and Hollo, Int. J. Pharm. 108: 69-75, 1994), liposomes (see , for example, Sharma et al., Cancer Res. 53: 5877-5881, 1993; Sharma and Straubinger, Pharm. Res. 11 (60): 889-896, 1994; WO 93/18751 and US Pat. . No. 5,242,073), liposome / gel (WO 94/2654), nanocapsules (Bartoli et al., J. Microencapsulation 7 (2): 191-197, 1990), micelles (Alkan-Onyuksel et al., Pharm Res. 11 (2): 206-212, 1994), implants (Jampel et al., Invest. Ophthalm. Vis. Science 34 (11): 3076-3083, 1993; Walter et al., Cancer Res. 54 : 22017-2212,1994), nanoparticles (Violante and Lanzafame PAACR), modified nanoparticles (US Patent No. 5,145,684), nanoparticles (surface modified) (US Patent No. No. 5,399,363), Taxol Emulsion / Solution (U.S. Patent No. 5,407,683), Micelle (Surfactant) (U.S. Patent No. 5,403,858), Synthetic Phospholipid Compounds (U.S. Patent No. US No. 4,534,899), Gas Carried Dispersion (US Patent No. 5,301,664), Liquid Emulsions, Foam, Spray, Gel, Lotion, Cream, Ointment, Dispersed Vesicles, Particles or Droplets solid or liquid aerosols, microemulsions (US Pat. No. 5,330,756), polymer shell (nano- and micro-capsule) (US Patent No. 5,439,686), compositions based on taxoids in a surfactant (US Patent No. 5,438 .072), emulsion (Tarr et al., Pharm Res. 4: 62-165, 1987), nanospheres (Hagan et al., Proc. Intern. Symp. Control Rel. Bioact. Mater, 22, 1995; Kwon et al. ., Pharm Res. 12 (2): 192.195; Kwon et al., Pharm Res. 10 (7): 970-974; Yokoyama et al., J. Contr. Rel. 32: 269-277, 1994; Gref et al., Science 263: 1600-1603, 1994; Bazile et al., J. Pharm. Sci. 84: 493,498, 1994) and implants (US Patent No. 4,882,168).

As discussed in more detail below, the therapeutic agents of the present invention, which are optionally incorporated into one of the carriers described herein to form a therapeutic composition, can be prepared and used to treat or prevent a wide variety of diseases.

Treatment or prevention of inflammatory diseases

As indicated above, the present invention provides means for treating or preventing inflammatory diseases of the respiratory tract, comprising the step of administering an anti-microtubule agent to a patient. Representative examples of such inflammatory diseases include, for example, nasal polyps or chronic sinusitis.

Other examples of inflammatory diseases include asthma, hypersensitivity pneumonitis, asbestosis, silicosis, and other forms of pneumoconiosis, chronic bronchitis, and chronic obstructive airway disease.

Chronic inflammatory diseases of the respiratory tract

Within other aspects of the invention, anti-microtubule agents (and compositions) can be used to treat or prevent diseases such as chronic inflammatory disease of the respiratory tract. In particular, the anti-microtubule agent can be administered to the site of inflammation (or potential site of inflammation) in order to treat the disease. Suitable anti-microtubule agents are discussed in detail above and include, for example, taxanes (e.g., paclitaxel and docetaxel), campothecin, eleutherobine, sarcodictins, epothiolones A and B, discodermolide, deuterium oxide (D2O), hexylene glycol ( 2-methyl-2,4-pentanediol), tubercidin (7-diazaadenosine), LY290181 (2-amino-4 (3-pyridyl) -4H-naphtho (1,2-b) pyrian-3-carbodinitrile), fluoride aluminum, ethylene glycol bis- (succinimidylsuccinate), glycine ethyl ester, monoclane anti-idiotypic antibodies, microtubule assembly favoring protein (taxol-like protein, TALP), hypotic-induced cell swelling

ES 2 207 451 T3 (190 mosmol / l), insulin (100 nmol / l) or glutamine (10 mmol / l), dynein binding, giberlin, XCHO1 (kinesin-like protein), lysophosphatidic acid, lithium ion, components of plant cell wall (eg, poly-L-Lysine and extensin), glycerol buffers, Triton X-100 microtubule stabilizing buffer, microtubule-associated proteins (eg, MAP2, MAP4, tau, large tau, ensconsin, factor elongation 1-alpha (EF-1a) and E-MAP-115), cellular entities (for example, histone H1, myelin basic protein and kinetochore), endogenous microtubule structures (for example, axonemal structures, plugs and lids of GTP), polypeptide-only stable tubule (for example, STOP145 and STOP220) and tension of mitotic forces, as well as any of the analogs and derivatives of any of the foregoing. These agents can be delivered, in certain embodiments, in the form of a composition together with a polymeric carrier, or in a liposome formulation as set forth in more detail both above and below. Within preferred embodiments of the invention, the agents or compositions may be administered intranasally, systemically, by inhalation, topically (eg, in the case of nasal polyps), or into the sinus cavities.

Asthma

In certain aspects of the invention, anti-microtubule agents can be used to treat or prevent asthma. Briefly, asthma is a condition characterized by recurrent episodes of airway obstruction that can resolve spontaneously or in response to treatment. Although its exact etiology is not known, the condition is an exaggerated bronchoconstrictive and inflammatory response to stimuli that affects 5% of the population. An effective anti-microtubule therapy for asthma would alter one or more of the pathological characteristics of the condition, such as reducing the infiltration and activity of inflammatory cells (T cells, mast cells or eosinophils), reducing the proliferation and thickening of the epithelium of the airways. respiratory tract, inhibit proliferation of smooth muscle cells and hypertrophy in the airway wall, decrease mucous secretion in the airway lumen, block the activity of inflammatory cytokines (IL-3, IL-4, IL-5, GMSF) that induce and perpetuate inflammation and inhibit hyperplasia and hypertrophy in the secretory glands of the airways.

Clinically, an effective anti-microtubule therapy for asthma would do one or more of the following: decrease the severity of symptoms, decrease the duration of exacerbations, increase the frequency and duration of disease remission periods, prevent fixed impairment and disability and prevent chronic progression of dyspnea, catarrh and wheezing; while improving hypoxia, FEV1 (forced expiratory volume in one second), resistance to airflow and respiratory hypocapnia / alkalosis and decreased V: Q (ventilation: perfusion) coincidence.

The anti-microtubule agent can be administered in any way to achieve the above end goals. Preferred methods of administration include inhaled (eg, metered dose inhaler) nebulizer, through an endotracheal tube, microparticle inhalation) and systemic treatments (intravenous, subcutaneous or intramuscular injection or oral preparation). Systemic treatment would be administered to patients with severe exacerbations in which inhaled therapy was not adequate. The lowest dose capable of producing a clinical or pathological improvement would be used. For example, for paclitaxel, systemic chronic low-dose therapy can be administered at 10 to 50 mg / m2.² every 4 weeks depending on the response; a high dose of "pulse" therapy can be administered at 50 to 250 mg / m² in the acutely ill patient. For inhaled therapy, 0.01% to 1% paclitaxel can be directly inhaled via the above-mentioned delivery vehicles / formulations. This would result in a supply of 1 to 50 mg / m² of paclitaxel directly into the respiratory tract. This dose would be titrated according to the response. Other anti-microtubule agents can be administered at equivalent doses adjusted for the potency and tolerance of the agent.

Chronic Pulmonary Obstruction Disease (COPD)

OCPD includes a variety of conditions (chronic bronchitis, asthmatic bronchitis, chronic obstructive bronchitis, and emphysema) that lead to chronic airway obstruction. These states can cause severe disability and are the fourth leading cause of death in the US Clinically, they are all characterized by dyspnea, colds, wheezing, and recurrent respiratory tract infections. Indications for the disease include decreased FEV1, increased residual volume, V: Q mismatch, and hypoxemia. Pathologically, there is an increased production of mucosa, hyperplasia of the mucous glands, increased activity of proteases (mainly elastase), inflammation of the airways, and destruction of the alveolar wall. Despite a wide range of etiologies (smoking being the most common), improvement of any of the above symptoms, indications, or pathological procedures would favorably affect the condition; therefore, an effective anti-microtubule therapy for COPD would alter at least one of the aforementioned. Treatment with an anti-microtubule agent would be administered as described above for asthma: inhaled paclitaxel would be provided at 1 to 50 mg / m² repeated as needed, and for systemic paclitaxel therapy 10 to 50 mg / m would be given² every 1 to 4 weeks in a chronic administration or 50 to 250 mg / m² delivered as "pulses" in the acutely ill patient. Other anti-microtubule agents would be administered at clinically equivalent doses.

Formulation and administration

As indicated above, the anti-microtubule agents of the present invention can be formulated in a variety of forms (e.g., microspheres, pastes, films, sprays, ointments, creams, gels, and

ES 2 207 451 T3 similar). Additionally, the compositions of the present invention may be formulated to contain more than one anti-microtubule agent, to contain a variety of additional compounds, or to have certain physical properties (e.g., elasticity, a particular melting point, or speed of specified release). In certain embodiments of the invention, the compositions can be combined in order to achieve a desired effect (for example, various microsphere preparations can be combined in order to achieve both rapid, slow, or prolonged release of one or more anti-agents. -microtubules).

Anti-microtubule agents can be administered alone or in combination with pharmaceutically or physiologically acceptable carriers, excipients or diluents. Generally, such vehicles should be non-toxic to the recipients at the dosages and concentrations employed. Ordinarily, the preparation of such compositions includes combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such like EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are examples of appropriate diluents.

As indicated above, the anti-microtubule agents, compositions or pharmaceutical compositions provided by the present invention can be prepared for administration by a variety of different routes including, for example, the topical route to a site of inflammation, oral, rectal, intracranial, intrathecal, intranasal, intraocular, intravenous, subcutaneous, intraperitoneal, intramuscular, sublingual and intravesicular. Other representative routes of administration include direct administration (preferably with ultrasound, CT, fluoroscopic, MRI, or endoscopic guidance) to the site of disease.

The therapeutic agents, therapeutic compositions, and pharmaceutical compositions provided by the present invention may be placed in containers together with a packaging material that provides instructions regarding the use of such materials. Generally, such instructions include a tangible expression describing the concentration of the reagents, as well as in certain embodiments, the relative amounts of excipient ingredients or diluents (eg, water, saline, or PBS) that may be necessary to reconstitute the anti-agent. -microtubules, the anti-microtubule composition or the pharmaceutical composition.

The following examples are offered by way of illustration, and not by way of limitation.

Examples

As discussed above, chronic inflammation is a procedure characterized by infiltration of tissues with white blood cells (macrophages, lymphocytes, neutrophils, and plasma cells), destruction of tissues by inflammatory cells and cell products (oxygen-reactive species, degradation enzymes of tissues such as matrix metalloproteinases) and repeated attempts at connective tissue replacement repair (angiogenesis and fibrosis).

In order to assess anti-microtubule agents for their ability to affect chronic inflammation on the following pathological / biological endpoints: (1) inhibition of the white blood cell response (macrophages, neutrophils and T cells) that initiates the inflammatory peel; (2) inhibition of hyperproliferation of mesenchymal cells (fibroblasts, synoviocytes, etc.) leading to the development of fibrosis and loss of organ function; (3) inhibition of matrix metalloproteinase production / activity causing tissue deterioration; (4) disruption of angiogenesis that increases the inflammatory response and provides the necessary metabolic support for the growth and development of fibrous tissue; and (5) all of this must be accomplished without substantial toxicity to normal parenchymal cells or hindering normal synthesis of matrix components (eg, collagen and proteoglycans).

As discussed in more detail below, the activity of microtubule stabilizing agents such as, for example, paclitaxel, has been examined in various tissues and inflammatory disease states. These agents demonstrate an ability to alter many of the parameters of the above diseases.

Example 1

Effect of anti-microtubule agents on neutrophil activity

The example describes the effect of anti-microtubule agents on the response of neutrophils stimulated with opsonized CPPD crystals or opsonized zymosan. As shown in the experimental models discussed below, anti-microtubule agents are strong inhibitors of particle-induced neutrophil activation as measured by chemiluminescence, superoxide anion production, and degranulation in response to plasma-opsonized zymosan or microcrystals.

A. Materials and methods

Hanks buffered saline (HBSS), pH 7.4 was used throughout this study. All chemicals

ES 2 207 451 T3 were purchased from Sima Chemical Co. (St. Louis, MO) unless otherwise stated. All experiments were performed at 37 ° C unless stated otherwise.

1. Preparation and characterization of crystals

CPPD (triclinic) crystals were prepared. The crystal size distribution was approximately 33% less than 10 µm, 58% between 10 and 20 µm, and 9% greater than 20 µm. Crystals prepared under the above conditions are pyrogen-free and crystals produced under sterile, pyrogen-free conditions produced the same magnitude of neutrophil response as crystals prepared under normal, non-sterile laboratory conditions.

two. Crystal opsonization and zymosan

All experiments studying neutrophil responses to crystals or zymosan in the presence of paclitaxel were performed using plasma opsonized CPPD or zymosan. The opsonization of crystals or zymosan was done with 50% heparinized plasma at a concentration of 75 mg of CPPD or 12 mg of zymosan per ml of 50% plasma. The crystals or zymosan were incubated with plasma for 30 minutes at 37 ° C and then washed in excess HBSS.

3. Neutrophil preparation

Neutrophils were prepared from freshly collected human citrated whole blood. Briefly, 400 ml of blood was mixed with 80 ml of 4% T500 dextran (Pharmacia LKB, Biotechnology AB Uppsala, Sweden) in HBSS and allowed to settle for 1 hour. Plasma was collected continuously and 5 ml was applied to 5 ml of Ficoll Paque (Pharmacia) in 15 ml polypropylene tubes (Coming, NY). After centrifuging at 500 g for 30 minutes, the neutrophil pellets were washed free of erythrocytes by 20 seconds of hypotonic shock. Neutrophils were resuspended in HBSS, kept on ice, and used for experiments within 3 hours. Neutrophil viability and purity was always greater than 90%.

Four. Incubation of neutrophils with anti-microtubule agents (a) Paclitaxel

A stock solution of 12 mM paclitaxel in dimethylsulfoxide (DMSO) was freshly prepared before each experiment. This stock solution was diluted in DMSO to provide paclitaxel solutions in the concentration range of 1 to 10 mM. Equal volumes of these diluted paclitaxel solutions were added to neutrophils at 5,000,000 cells per ml under gentle centrifugation to achieve concentrations of 0 to 50 µΜ with a final DMSO concentration of 0.5%. Cells were incubated for 20 minutes at 33 ° C and then for 10 minutes at 37 ° C before addition to crystals or zymosan.

(b) Aluminum fluoride

A 1M aluminum fluoride (AlF3) stock solution in HBSS was recently prepared. This stock solution was diluted in HBSS to provide AlF3 solutions in the concentration range of 5 to 100 mM. Equal volumes (50 μ ^ of these dilute solutions of Alf₃ to neutrophils at 5,000,000 cells per ml incubated for 15 minutes at 37 ° C. Luminol (1 µΜ) was added followed by 20 µl opsonized zymosan (final concentration = 1 mg / ml) to activate the cells.

(c) Glycine ethyl ester

A 100 mM glycine ethyl ester stock solution in HBSS was recently prepared. This stock solution was diluted in HBSS to provide glycine ethyl ester solutions in the concentration range of 0.5 to 10 mM. Equal volumes (50 µ ^ of these diluted glycine ethyl ester solutions were added to neutrophils at 5,000,000 cells per ml and incubated for 15 minutes at 37 ° C. Luminol (1 µΜ) was added followed by 20 µl opsonized zymosan (final concentration = 1 mg / ml) to activate the cells.

(d) LY290181

A stock solution of LY290181 was recently prepared at 100 µΜ in HBSS. This stock solution was diluted in HBSS to provide LY290181 solutions in the concentration range of 0.5 to 50 µΜ. Equal volumes (50 μ ^ of these diluted LY290181 solutions were added to neutrophils at 5,000,000 cells per ml and incubated for 15 minutes at 37 ° C. Luminol (1 μΜ) was added followed by 20 μl of opsonized zymosan ( final concentration = 1 mg / ml) to activate the cells.

ES 2 207 451 T3

5. Chemiluminescence Assay

All chemiluminescence studies were performed at cell concentrations of 5,000,000 cells / ml in HBSS with CPPD (50 mg / ml). In all experiments 0.5 ml of cells were added to 25 mg of CPPD or 0.5 mg of zymosan in 1.5 ml of capped Eppendorf tubes. 10 µl of luminol dissolved in 25% DMSO in HBSS was added to a final concentration of 1 M and the samples were mixed to initiate neutrophil activation by the zymosan crystals. Chemiluminescence was verified using an LKB luminometer (model 1250) at 37 ° C for 20 minutes with shaking immediately prior to measurements to resuspend crystals or zymosan. Control tubes contained cells, drug, and luminol (crystals were absent).

6. Generation of superoxide anion

Superoxide anion concentrations were measured using the inhibible superoxide dismutase reduction of the cytochrome C assay. Briefly, 25 mg of crystals or 0.5 mg of zymosan were placed in a 1.5 ml capped Eppendorf tube and heated. at 37 ° C. 0.5 ml of cells were added at 37 ° C along with ferricitochrome C (final concentration 1.2 mg / ml) and the cells were activated by turning the capped tubes. At the appropriate times, the tubes were centrifuged at 10,000 g for 10 seconds and the supernatant was collected to be assayed by measuring the absorbance at 550 nm. The control tubes were constituted under the same conditions, with the inclusion of superoxide-dismutase at 600 units per ml.

7. Enutrophil Degranulation Assay

1 1/2 millimeter Eppendorf tubes containing 25 mg of CCPPD or 1 mg of zymosan were preheated to 37 ° C. 0.5 ml of cells were added at 37 ° C and then vigorously shaken to initiate reactions. At the appropriate times, the tubes were centrifuged at 10,000 g for 10 seconds and 0.4 ml of the supernatant was stored at -20 ° C for further testing.

The lysozyme was tested by the decrease of the absorbance at 450 nm of a suspension of Micrococcus lisodeikticus. Briefly, Micrococcus lisodeikticus was suspended at 0.1 mg / ml in 65 mM potassium phosphate buffer, pH 6.2, and the absorbance at 450 nm was adjusted to 0.7 units by dilution. The crystal (or zymosan) and the supernatant cell matter (100 μ ^ were added to 2.5 ml of the Micrococcus suspension and the decrease in absorbance was verified. Lysozyme standards (chicken egg white) in the range of 0 to 2,000 units / ml were prepared and a calibration plot of lysozyme concentration versus rate of decrease in absorbance at 450 nm was obtained.

Myeloperoxidase (MPO) activity was measured by the increase in absorbance to 450 mm that accompanies the oxidation of dianisidine. 7.8 mg of dianisidine was dissolved in 100 ml of 0.1 M citrate buffer, pH 5.5, at 3.2 mM, by sonication. 0.89 ml of the dianisidine solution were added to a 1 ml cuvette, followed by 50 μl of 1% Triton x 100, 10 μl of 0.05% hydrogen peroxide in aqueous solution and 50 μl of supernatant of crystals-cells. MPO activity was determined from the change in absorbance (450 nm) per minute, Delta 450, using the following equation:

Dianisidine oxidation (nmol / min) = 50 x Delta A 450

8. Neutrophil viability

To determine the effect of anti-microtubule agents on neutrophil viability, the release of the cytoplasmic marker enzyme, lactate dehydrogenase (LDH) was measured. Control tubes containing drug cells (in the absence of crystals) were also tested for LDH from degranulation experiments.

B. Results

Statistical significance was determined in all experiments using the Students' t-test and significance was claimed at p <0.05. When error bars are displayed, they describe a standard deviation around the mean value for the given number n.

1. Neutrophil viability (a) Paclitaxel

Neutrophils treated with paclitaxel at 46 AM for one hour at 37 ° C did not show any increased level of LDH release (always less than 5% of the total) above controls, indicating that paclitaxel did not cause cell death.

(b) Aluminum fluoride

Neutrophils treated with aluminum fluoride at a concentration range of 5 to 100 mM for 1 hour at

ES 2 207 451 T3

37 ° C did not show any increased level of LDH release above controls, indicating that aluminum fluoride did not cause cell death.

(c) Glycine ethyl ester

Neutrophils treated with glycine ethyl ester at a concentration range of 0.5 to 20 mM for 1 hour at 37 ° C did not show any increased level of LDH release above controls indicating that glycine ethyl ester did not cause cell death.

two. Chemiluminescence (a) Paclitaxel

Paclitaxel at 28 μΜ produced a strong inhibition of neutrophil chemiluminescence induced by both plasma-opsonized CPPD and plasma-opsonized zymosan, as shown in Figures 1A, 1B and 2A, respectively. The inhibition of the peak chemiluminescence response was 52% (+/- 12%) and 45%. % (+/- 11%) for CPPD and zymosan, respectively. Inhibition by paclitaxel at 28 µΜ of chemiluminescence induced by both plasma opsonized CPPD and plasma opsonized zymosan was significant for all times from 3 to 16 minutes (Figures 1 and 4A). Figures 1A and 1B show the concentration dependence of paclitaxel inhibition of plasma opsonized CPPD-induced neutrophil chemiluminescence. In all experiments, the control samples never produced chemiluminescence values ​​greater than 5 mV and the addition of paclitaxel at all concentrations used in this study had no effect on the chemiluminescence values ​​of the controls.

(b) Aluminum fluoride

Aluminum fluoride at concentrations of 5 to 100 mM produced strong inhibition of plasma opsonized zymosan-induced neutrophil chemiluminescence, as shown in Figure 1C. This figure shows the concentration dependence of AlF3 inhibition of plasma opsonized zymosan-induced neutrophil chemiluminescence. The addition of AlF3 at all concentrations used in this study had no effect on the chemiluminescence values ​​of the controls.

(c) Glycine ethyl ester

Glycine ethyl ester at concentrations of 0.5 to 20 mM produced strong inhibition of plasma opsonized zymosan-induced neutrophil chemiluminescence, as shown in Figure 1D. This figure shows the concentration dependence of glycine ethyl ester inhibition of plasma opsonized zymosan-induced neutrophil chemiluminescence. The addition of glycine ethyl ester at all concentrations used in this assay had no effect on the chemiluminescence values ​​of the controls.

(d) LY290181

LY290181 at concentrations of 0.5 to 50 µΜ produced strong inhibition of plasma opsonized zymosan-induced neutrophil chemiluminescence, as shown in Figure 1E. This figure shows the concentration dependence of LY290181 inhibition of plasma opsonized zymosan-induced neutrophil chemiluminescence. The addition of LY290181 at all concentrations used in this study had no effect on the chemiluminescence values ​​of the controls.

3. Generation of superoxides

The time course of the production of superoxide anions induced by plasma opsonized CPPD crystals, as measured by the inhibible reduction of superoxide dismutase (SOD) of cytochrome C is shown in Figure 3. Treatment of cells with paclitaxel a 28 μΜ produced a decrease in the amount of superoxide generated for all time. This decrease was significant for all times shown in Figure 3A. The concentration dependence of this inhibition is shown in Figure 3B. Stimulation of superoxide anion production by opsonized zymosan (Figure 4B) showed a time course similar to CPPD-induced activation. The zymosan-induced superoxide anion production by paclitaxel at 28 µΜ was less considerable than the inhibition of activation by CPPD, but was significant for all times shown in Figure 4B.

Treatment of CPPD crystal-induced neutrophils with LY290181 at 47 μΜ also produced a decrease in the amount of superoxide generated (Figure 3C).

Four. Neutrophil degranulation

Neutrophil degranulation was verified by crystal-induced release of CPPD opsonized with myeloperoxidase plasma and lysozyme or the release induced by zymosan opsonized with myeloperoxidase plasma.

ES 2 207 451 T3

Sufficient amounts of these two enzymes have been shown to be released into extracellular media when plasma-coated CPPD crystals are used to stimulate neutrophils without the need to add cytochalasin B to cells. Figures 5 and 2 show the time course of MPO and lysozyme release, respectively, from plasma-coated CPPD-stimulated neutrophils. Figure 5A shows that paclitaxel inhibits myeloperoxidase release from neutrophils activated by plasma opsonized CPPD in the first 9 minutes of crystal-cell incubation. Paclitaxel significantly inhibited CPPD-induced myeloperoxidase release for all times, as shown in Figure 5A. Figure 5B shows the concentration dependence of paclitaxel inhibition of CPPD-induced myeloperoxidase release:

Paclitaxel at 28 μΜ reduced lysozyme release, and this inhibition of degranulation was significant for all times, as shown in Figure 2.

Only minor amounts of MPO and lysozyme were released when neutrophils were stimulated with opsonized zymosan. Despite these low levels, it was possible to verify a 50% inhibition of MPO release after a 9-minute incubation in the presence of paclitaxel at 28 μΜ, but it was statistically significant (p <0.05) (not shown the data). Treatment of CPPD crystal-induced neutrophils with LY290181 at 17 µΜ decreased the release of both lysozyme and myeloperoxidase from cells (Figures 5C and 5D).

C. Explanation

These experiments demonstrate that paclitaxel and other anti-microtubule agents are strong inhibitors of crystal-induced neutrophil activation. Furthermore, by showing similar levels of inhibition in neutrophil responses to another form of particulate activator, opsonized zymosan, it is clear that the inhibitory activity of paclitaxel and other anti-microtubule agents is not limited to neutrophil responses to crystals. Paclitaxel, aluminum fluoride, glycine ethyl ester, and LY290181 were also shown to be strong inhibitors of zymosan-induced neutrophil activation without causing cell death. LY290181 was shown to decrease superoxide anion production and CPDD crystal-induced neutrophil degranulation.

Example 2

T cell response to an antigenic stimulus

In order to determine whether paclitaxel affects T cell activation in response to stimgenes, TR1 T cell clones were stimulated with myelin basic protein peptide, GP68-88, or lectin, with A, for 48 hours. in the absence or presence of increasing concentrations of paclitaxel in a micellar formulation. Paclitaxel was added at the beginning of the experiment or 24 hours after stimulation of the cells with peptide or A. Tritiated thymidine incorporation was determined as a measure of T cell proliferation in response to stimulation with peptides or A.

The results demonstrated that T cell stimulation increased in response to the GP68-88 peptide and with A. In the presence of control polymer micelles, the T cell stimulation in response to both agonists was not altered. However, treatment with paclitaxel micelles, either at the beginning of the experiment or 24 hours after challenge, decreased the T-cell response in a concentration-dependent manner. Under both conditions, T cell proliferation was completely inhibited by 0.02 µΜ paclitaxel (Figure 79).

These data indicate that paclitaxel is a potent inhibitor of T cell proliferation in response to antigen-induced stimulation.

Example 3

Effect of paclitaxel on synoviocyte cell proliferation in vitro

The experiments were performed in order to assess the effect of different concentrations of paclitaxel on tritiated thymidine incorporation (a measure of synoviocyte DNA synthesis) and in vitro cell proliferation.

A. Materials and methods

1. Incorporation of H³-thymidine in synoviocytes

Synoviocytes were incubated with different concentrations of paclitaxel (10^-5 Μ, 10^-6 Μ, 10^-7 M and 10^-8 M) continuously for 6 or 24 hours in vitro. At these time values, 1 x 10 was added^-6 cpm of H³-thymidine to cell culture and incubated for 2 hours at 37 ° C. The cells were placed through a cell collector, washed through a filter, the filters were cut, and the amount of radiation contained in the filter sections was determined. Once the amount of thymidine incorporated into the cells was determined, it was used to determine the rate of cell proliferation. This experiment was repeated three times and the data was compared.

ES 2 207 451 T3

two. Synoviocyte proliferation

Bovine synovial fibroblasts were grown in the presence and absence of different concentrations (10^-5 M, 10^-6 M, 10^-7 M and 10^-8 M) of paclitaxel for 24 hours. At the end of this time period, the total number of visually viable synoviocyte cells was determined by counting by dye exclusion using trypan blue staining. This experiment was performed 4 times and the data was collected.

B. Results

1. Incorporation of H³ -thymidine in synoviocytes

This study demonstrated that paclitaxel at low concentrations inhibits the incorporation of H³-thymidine (and by extension DNA synthesis) in synoviocytes at concentrations as low as 10^-8 M. At six hours there was no significant difference between the degree of inhibition produced by the higher versus lower concentrations of paclitaxel (Figure 8). However, within 24 hours part of the effect was lost at the lower concentrations of the drug (10-8 M), but was still substantially lower than that observed in control animals.

two. Synoviocyte proliferation

This study demonstrated that paclitaxel was cytotoxic to the proliferation of synovial fibroblasts in a concentration-dependent manner. Paclitaxel at concentrations as low as 10^-7 M is capable of inhibiting the proliferation of synoviocytes (Figure 9). At higher concentrations of paclitaxel (10^-6 M and 10^-5 M) the drug was toxic to synovial fibroblasts in vitro.

C. Explanation

The above study demonstrates that paclitaxel is capable of inhibiting the proliferation of synovial-derived fibroblasts at relatively low concentrations in vitro. Therefore, given the role of connective tissue in the development of chronic inflammation and its behavior during the pathogenesis of inflammatory disease, blocking cell proliferation will favorably affect the outcome of the disease in vivo.

Example 4 (Reference example)

Characterization of the activity of paclitaxel on human epidermal keratinocytes in vitro

The time and dose dependent effects of paclitaxel on actively proliferating human keratinocytes and HaCAT keratinocytes (spontaneously immortalized human epidermal keratinocytes) were investigated.

A. Materials and methods

The effect of paclitaxel on keratinocytes was assessed by determining the number of cells and the incorporation of H³-thymidine by cells. For thymidine incorporation, low-density plating keratinocytes (in DMEM, supplemented with 10% FCS, glutamine, antibiotics) were treated with paclitaxel concentrations from 0 to 10^-4 M for 6 hours during logarithmic growth. The H³ -thymidine was added to the cells and incubated for an additional 6 hours. Cells were harvested and radioactivity was determined. To determine total cell numbers, keratinocytes were plated as described and incubated in the presence and absence of paclitaxel for 4 days. After incubation, cells were harvested and counted by trypan blue exclusion assay.

B. Results

The number of viable cells was determined as a percentage of untreated controls. At a paclitaxel concentration of 10^-9 M, cell viability was greater than 100% of untreated controls, while at a viability of 10^-8 M, the viability was slightly less than 87% (Figure 7). There was a significant drop in cell viability at a paclitaxel concentration of 10^-7 or older.

C. Explanation

Paclitaxel was extremely cytotoxic to human keratinocytes at concentrations as low as 10^-7M. In psoriasis, keratinocytes are abnormally proliferating cells, and since paclitaxel stabilizes microtubules, its effect on this mitotically active system is to be expected. In other studies, paclitaxel was found to be cytotoxic to proliferating synoviocytes, but had no effect on non-proliferating chondrocytes. Therefore, paclitaxel can act on hyperproliferative cells in psoriatic lesions, while it is non-toxic for normal epidemic cells.

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Example 5 (Reference Example)

Effect of paclitaxel on astrocyte proliferation

It is well established that there is an increase in the numbers of fibrous astrocytes in MS lesions, which are believed to be involved in the destruction of myelin through the production of matrix cytokines and metalloproteinases (Mastronardi et al., J. Neurosci Res. 36: 315-324, 1993; Chandler et al., J. Neuroimmunol. 72: 155-161, 1997). Fibrous astrocytes have elevated levels of glial fibrillar acidic protein (GFAP) that serves as a biochemical marker for fibrous astrocyte proliferation. The ability of paclitaxel micelles to inhibit astrocyte proliferation was assessed in a transgenic mouse model of demyelinating disease (Mastronarci et al., J. Neurosci. Res. 36: 315-324, 1993).

A. Materials and methods

Subcutaneous administration of a continuous therapy of paclitaxel (2 mg / kg; 3 x week, total of 10 injections) was started at the clinical onset of the disease (approximately 4 months time). Five animals received micellar paclitaxel, two mice were used as controls; one mouse was an untreated normal one and one was an untreated transgenic specimen. Only a transgenic mouse was used as a control because the course of the disease has been well established in the laboratory. Four-month-old animals were injected with micellar paclitaxel after initial signs of the neurological pathology of MS were evident.

Three days after the 10th injection, the experimental study was terminated and brain tissues were treated for histological analysis. For the light microscope, the tissues were fixed in formalin and embedded in paraffin. Sections were stained with anti-GFAP antibody (DACO), washed, and then reacted with HPP-conjugated secondary antibody. Sections were stained for HPP and counterstained with hematoxylin. For the electron microscope, the tissues were fixed in 2.5% glutaraldehyde and phosphate buffered saline (pH 7.2) and subsequently fixed with 1% osmium tetroxide. Sections were prepared and viewed with a JEOL 1200 EX II transmission electron microscope.

B. Results

As neurological pathology progresses, GFAP levels rise in the brains of transgenic mice. This is believed to reflect an increase in the number of fibrous astrocytes present. In contrast, transgenic mice treated with paclitaxel had almost normal levels of GFAP (Table 1). These data suggest that paclitaxel may inhibit astrocyte proliferation in vivo, which may contribute to the prevention of demyelination in MS.

<td colspan="3">TABLE 1</td>

<td>Quantification</td><td colspan="2">of GFAP in brain homogenate</td>

<td>Group</td><td>GFAP (ng)</td><td>GFAP (ng / ^ g protein homogenate)</td>

<td>Normal mice Transgenic mice</td><td> 0,64 + 0,02</td><td> 12,8</td>

<td>unique Transgenic mice only treated with</td><td> 1,80 + 0,10</td><td> 36,0</td>

<td>paclitaxel</td><td> 0,69 + 0,05</td><td> 13,8</td>

ES 2 207 451 T3

An additional analysis of GFAP in brain tissue was assessed histologically. Figure 78 illustrates brain sections from normal mice, control transgenic mice not treated with paclitaxel, and transgenic mice treated with paclitaxel.

Although control transgenic mice have higher numbers of fibrous astrocytes, the morphology of the astrocytes is similar to that observed in normal animals (coarse stellate procedures extending from the cell structure). However, in transgenic mice treated with paclitaxel, the number of fibrous astrocytes was significantly decreased. Additionally, two morphological changes were observed: the cellular structure of the fibrous astrocytes appears to round out (which has been shown to lead to apoptosis in culture) and the cellular processes become very fine around the cell structure.

Further ultrastructural analysis using electron microscopy has shown that transgenic mouse astrocytes were characterized by densely stained astrocytic processes originating from cell structure. These broad procedures contain a well-organized arrangement of filaments indicating a viable activated cell. However, astrocyte morphology in paclitaxel-treated transgenic animals was characterized by cell rounding, fine filamentous procedures, and intracellular depletion and disorganization of filamentous proteins (Figure 80).

C. Conclusions

These data demonstrate that paclitaxel causes changes relative to fibrous astrocytes in vivo, the most proliferating cell type in MS lesions. It is likely that paclitaxel is also inhibiting the function of astrocytic procedures and, therefore, may alter the cellular events involved in the destruction of myelin.

Example 6

Effect of paclitaxel on endothelial cell proliferation

In order to determine whether paclitaxel inhibits endothelial cell proliferation, EOMA cells (an endothelial cell line) were placed at low density and incubated in the absence and presence of increasing concentrations of paclitaxel for 48 hours. After incubation, the number of viable cells was determined using the trypan blue exclusion assay. The results (provided in Figure 9) show that paclitaxel at concentrations of 10^-8 M inhibited endothelial cell proliferation above 50% and at concentrations of 10^-7 M or greater cell proliferation was completely inhibited. These data demonstrate that paclitaxel is a potent inhibitor of endothelial cell proliferation. Cell toxicity tests were performed three times, and each individual measurement was done in triplicate.

In order to determine the effect of paclitaxel on endothelial cell cycling and apoptosis, EOMA cells were incubated in the absence and presence of increasing concentrations of paclitaxel for 24 hours. Cells were fixed with 3.7% formaldehyde in phosphate buffered saline for 20 minutes and stained with DAPI (4 ', 6-diamidino-2-phenylindole), 1 μg / ml and examined with a low 40x objective. epifluorescent optical instruments. Apoptotic cells were evaluated by titrating the cells for fragmented nuclei and condensed chromatin. The data show that paclitaxel concentrations greater than 10^-8 M induced endothelial cell apoptosis (Figure 10).

Example 7

Proliferation Assay Protocol (MTT)

On the first day, 5-10 x 10⁴ Synoviocytes were plated per well (96-well plate). Column # 1 was kept cell free (blank test). On day 2, the plate was flipped to discard the medium and 200 µl of medium containing various concentrations of drug was added. Cells were exposed for 6 hours, 24 hours, or 4 days. No drug was added to columns # 1 and # 2 (blank and untreated control, respectively). The medium containing the drug was discarded and 200 µl of fresh complete medium was added. The cells were then allowed to grow for an additional 3 to 4 days. On day five, 200 µl of dimethylthiazoldiphenyltetrazolium bromide (MTT) salt (5 mg / ml PBS) was added and allowed to incubate for 4 hours at 37 ° C. The medium was pelleted and 200 µl of DMSO was added. The Plate was shaken for 30 minutes and the absorbance was read at 562 nm.

Results

The data were expressed as the% survival that was obtained by dividing the number of cells that remained after treatment by the number of cells in the untreated control column No. 2 (the number of cells was obtained from a pattern made before of the test). The IC50, the drug concentration that kills 50% of the population, can be interpolated from Figures 1A-E. During a 24 hour exposure, the compound LY290181 was found to be the most potent anti-microtubule agent for reducing and inhibiting cell proliferation, with an IC50 of less than 5 nM (Figure C). Paclitaxel, epothilone B, and tubercidin were slightly less potent with IC50s

ES 2 207 451 T3 of approximately 30 nM (Figure A), 45 nM (Figure F) and 45 nM (Figure B), respectively. Finally, the IC50s for aluminum fluoride (AlF3) and hexylene glycol were significantly higher, with values ​​of approximately 32 μΜ (Figure E) and 64 mM (Figure D), respectively.

Example 8

Effect of paclitaxel and other anti-microtubule agents on matrix metalloproteinase production

A. Materials and methods

1. IL-1 Stimulated AP-1 Transcriptional Activity Inhibited by Paclitaxel

Chondrocytes were transfected with constructs containing a CAT reporter gene driven by AP-1, and stimulated with IL-1, IL-1 (50 ng / ml) was added and incubated for 24 hours in the absence and presence of paclitaxel at various concentrations . Paclitaxel treatment decreased CAT activity in a concentration-dependent manner (mean + SD). The data marked with an asterisk (*) are significant compared to the CAT activity induced by IL-1 according to a t-test, P &lt;0.05. The results shown are representative of three independent experiments.

two. Effect of paclitaxel on IL-1 induced AP-1 DNA binding activity, AP-1 DNA

Binding activity was tested with a radiolabeled human AP-1 sequence probe and a gel mobility shift assay. Untreated chondrocyte extracts or treated with various amounts of paclitaxel (10^-7 to 10^-5 M) followed by IL 1β (20 ng / ml) were incubated with excess probe on ice for 30 minutes and then by non-denaturing gel electrophoresis. The "com" string contains excess unlabeled AP-1 oligonucleotide. The results shown are representative of three independent experiments.

3. Effect of paclitaxel on IL-1-induced MMP-1 and MMP-3 mRNA expression

Cells were treated with paclitaxel at various concentrations (10^-7 to 10^-5 M) for 24 hours. They were then treated with IL-1β (20 ng / ml) for an additional 18 hours in the presence of paclitaxel. Total RNA was isolated, and MMP-1 mRNA levels were determined by Northern blot analysis. The blots were then withdrawn and reexamined with P-radiolabeled rat GAPDH-cDNA.³², which was used as a house gene. The results shown are representative of four independent experiments. Quantification of collagenase-1 and stromelysin expression mRNA levels. The expression levels of MP-1 and MMP-3 were normalized with GAPDH.

Four. Effect of other anti-microtubules on collagenase expression

Primary chondrocyte cultures were recently isolated from calf cartilage. Cells were plated at 2.5 x 10⁶ per ml in 100 x 200 mm culture dishes and incubated in Ham's F12 medium containing 5% FBS overnight at 37 ° C. Cells were starved in serum-free medium overnight and then treated with anti-microtubule agents at various concentrations for 6 hours. IL-1 (20 ng / mg) was then added to each plate and the plates were incubated for an additional 18 hours. Total RNA was isolated by the acidified guanidine isothiocyanate method and electrophoresed on a denatured gel. Denatured RNA samples (15 μβ) were analyzed by gel electrophoresis on 1% denatured gel and transferred to a nylon membrane and hybridized with the P-labeled collagenase-cDNA probe.³². Glyceraldehyde phosphate dehydrase (GAPDH) -cDNA labeled with P³² it is an internal standard to ensure approximately equal load. The exposed films were scanned and quantitatively analyzed with ImageQuant.

B. Results

1. Promoters on the matrix metalloproteinase group

Figure 19A shows that all matrix metalloproteinases contained the transcriptional elements AP-1 and PEA-3, with the exception of Gelatinase B. It has been well established that the expression of matrix metalloproteinases such as collagenases and stromelysins is activation dependent. of AP-1 transcription factors. Thus, AP-1 inhibitors will inhibit the expression of matrix metalloproteinases.

two. Effect of paclitaxel on AP-1 transcriptional activity

As shown in Figure 19B, IL-1 stimulated the transcriptional activity of AP-1 5-fold. Pretreatment of transiently transfected chondrocytes with paclitaxel reduced the CAT activity of the IL-1-induced AP-1 reporter gene. Thus, IL-1-induced AP-1 activity was reduced in chondrocytes by paclitaxel in a concentration-dependent manner (10^-7 to 10^-5 M). These data demonstrated that paclitaxel was a potent inhibitor of AP-1 activity in chondrocytes.

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3. Effect of paclitaxel on AP-1 DNA binding activity

To confirm that paclitaxel inhibition of AP-1 activity was not due to non-specific effects, the effect of paclitaxel on IL-1-induced AP-1 binding to oligonucleotides was examined using chondrocyte nuclear lysates. As shown in Figure 19C, IL-1-induced binding activity was decreased in chondrocyte lysates that had been pretreated with paclitaxel at a concentration of 10^-7 to 10^-5 Μ for 24 hours. Paclitaxel inhibition of AP-1 transcriptional activity was closely correlated with decreased AP-1 DNA binding.

Four. Effect of paclitaxel on the expression of collagenase and stromelysin

As paclitaxel was a potent inhibitor of AP-1 activity, the effect of paclitaxel or IL-1-induced expression of collagenase and stromelysin, two important matrix metalloproteinases involved in inflammatory diseases, was examined. Briefly, as shown in Figure 20, IL-1 induction increases the levels of collagenase and stromelysin mRNA in chondrocytes. Pretreatment of chondrocytes with paclitaxel for 24 hours significantly reduced collagenase and stromelysin mRNA levels. For a paclitaxel value of 10^-5 Μ, there was a complete inhibition. The results show that paclitaxel completely inhibited the expression of two matrix metalloproteinases at concentrations similar to those that inhibit AP-1 activity.

5. Effect of other anti-microtubules on collagenase expression

Figures 12A-H demonstrate that anti-microtubule agents inhibited collagenase expression. Collagenase expression was stimulated by the addition of IL-1, which is a pro-inflammatory cytokine. Preincubation of chondrocytes with various anti-microtubule agents, specifically LY290181, hexylene glycol, deuterium oxide, glycine ethyl ester, AlF3, tubercidin-epothilone, and ethylene glycol bis (succinimidylsuccinate), prevented induced collagenase expression in all cases by IL-1 at concentrations as low as 1 x 10^-7 Μ.

C. Explanation

Paclitaxe l was able to inhibit the expression of collagenase and stromelysin in vitro at concentrations of 10^-6Μ. As this inhibition can be explained by inhibition of AP-1 activity, a required step in the reduction of all matrix metalloproteinases with the exception of gelatinase B, it is expected that paclitaxel inhibits other matrix metalloproteinases that are dependent by AP-1. Levels of these matrix metalloproteinases are elevated in all inflammatory diseases and play a major role in matrix degradation, cell displacement and proliferation, and angiogenesis. Therefore, inhibition by paclitaxel of the expression of matrix metalloproteinases such as collagenase and stromelysin will have an advantageous effect in inflammatory diseases.

Example 9

Effect of anti-microtubule agents on the expression of proteoglycans

Primary chondrocyte cultures isolated from calf cartilage were recently isolated. Cells were plated at 2.5 x 10^-6 per ml in 100 x 200 mm culture dishes and incubated in Ham's F12 medium containing 5% FBS overnight at 37 ° C. Cells were starved in serum-free medium overnight and then treated with anti-microtubule agents at various concentrations.^-17 Μ, 10^-6 Μ, 10^-5 Μ and 10^-4 Μ) for 6 hours. IL-1 (20 ng / ml) was then added to each plate and the plates were incubated for an additional 18 hours. Total RNA was isolated by the acidified guanidine isothiocyanate method and electrophoresed on a denatured gel. Denatured RNA samples (15 μβ) were analyzed by gel electrophoresis on a 1% denatured gel, transferred to a nylon membrane, and hybridized with the P-labeled proteoglycan cDNA probe.³² (aggrecan). The P-labeled glyceraldehydephosphate dehydrase cDNA³² (GAPDH) is an internal standard to ensure approximately equal load. The exposed films were scanned and quantitatively analyzed with ImageQuant.

Results

Figures 13A-H show that anti-microtubule agents that had an inhibitory effect on collagenase expression (Example 8), specifically LY290181, hexylene glycol, deuterium oxide, glycine ethyl ester, AlF3, turbecidin-epothilone, and bis (succinimidylsuccinate) of ethylene glycol, did not affect the expression of aggrecan, a main component of the cartilage matrix, at all concentrations evaluated.

Example 10

NF-kB activity assay (cell-based)

IL-1 and TNF were both identified as pro-inflammatory cytokines that activate the transcription of genes driven by a transcription factor called Nf-kB, also involved in inflammatory procedures23

ES 2 207 451 T3 tories. Therefore, the inflammatory effect of IL-1 and TNF can be indirectly assessed by means of a reporter gene assay (NF-kB) that responds to IL-1 and TNF stimulation.

On the first day, 5 x 10⁴ NIH-3T3 (murine fibroblast), stably transfected with NF-kB reporter construct (Luciferase, Promega Corp.), was plated per well (24-well plate). Once confluent (on days 3-4), cells were starved by replacing complete medium with 1 ml of serum-free medium. After 24 hours of starvation, cells were treated with various concentrations of anti-microtubule agents 6 hours before the addition of IL-1 (20 ng / ml) and TNF (20 ng / ml). Cells were exposed to IL-1 and TNF for 1 hour and 16 hours and NF-kB activity was measured 24 hours later. On the fifth day, the medium was discarded and the cells were rinsed once with PBS. The cells were then extracted for 15 minutes with 250 µl of lysis buffer (Promega Corp., Wisconsin). The transcriptional activity of NF-kB was assessed by adding 25 µl of luciferase substrate to a tube containing 2.5 µl of cell extract. The tube was immediately inserted into a luminometer (Turner Designs) and the light emission was measured for 10 seconds. Luciferase data were then normalized for protein concentration.

Results

The data were expressed by showing the interference that the anti-microtubule agents exhibited on the induction of NF-kB (multiplied induction). As shown in Figures 80A, 80B, 80C, and 80D, tubercidin and paclitaxel inhibited both IL-1 and TNF-induced NF-kB activity. The inhibitory effect of tubercidin and paclitaxel for the 6-hour and 24-hour treatments was approximately 10 µM and 2 µM, respectively. Example 11

Inhibition of tumor angiogenesis by paclitaxel

Fertilized domestic chicken embryos are incubated for 4 days before removing their shells. The egg contents are emptied by removing the shell placed around the airspace, breaking the inner shell membrane, piercing the opposite end of the shell, and allowing the egg contents to slide smoothly out of the blunt end. The contents are poured into sterilized round bottom glass bowls covered with petri dish lids and incubated at 90% relative humidity and 3% carbon dioxide.

MDAY-D2 cells (a murine lymphoid tumor) are injected into mice and allowed to grow into tumors weighing 0.5-1.0 g. Mice are euthanized, tumor sites are rubbed with alcohol, excised, placed in sterile tissue culture media, and sliced ​​into 1 mm pieces under a laminar flow hood. Before placing the dissected tumors in the 9-day-old chick embryos, the CAM surfaces are gently scraped with a 30-gauge needle to ensure implantation of the tumors. The tumors are then placed on the CAMs after 8 days of incubation (4 days after peeling) and allowed to grow on the CAMs for four days to establish vascular supply. Four embryos are prepared using this method, each embryo receiving 3 tumors. On day 12, each of the 3 tumors in the embryos received thermopaste containing 20% ​​paclitaxel, thermopaste with no content, or no treatment. The treatments were continued for two days before the results were recorded.

The explanted MDAY-D2 tumors secrete angiogenic factors that induce ingrowth of capillaries (derived from CAM) into the tumor mass and this is allowed to continue to grow in size. As all tumor vessels derive from the CAM, although all tumor cells derive from the explant, it is possible to assess the effect of therapeutic interventions on these two procedures independently. This assay has been used to determine the efficacy of paclitaxel-containing thermopaste on: (a) inhibiting tumor vascularization and (b) inhibiting the growth of tumor cells themselves.

Direct in vivo stereomicroscopic evaluation and histological examination of the fixed tissues from this study demonstrated the following. In tumors treated with thermopaste containing 20% ​​paclitaxel, there was a reduction in the number of blood vessels supplying the tumor (Figures 14C and 14D), a reduction in the number of blood vessels within the tumor, and a reduction in the number of blood vessels in the periphery of the tumor (the area that is typically the most highly vascularized in a solid tumor) when compared to control tumors (Figures 14A and 14B). The tumors began to shrink in size and mass during the two days the study was conducted. Additionally, it was observed that numerous endothelial cells were arrested in cell division, indicating that endothelial cell proliferation had been affected. Tumor cells were also observed to frequently arrest in mitosis. All 4 embryos showed a consistent pattern that thermopaste containing 20% ​​paclitaxel suppressed tumor vascularization, while thermopaste without content had no effect.

In comparison, in CAMs treated with empty thermopaste, the tumors were well vascularized, with an increase in the number and density of vessels, compared to that of the normal surrounding tissue, and considerably more vessels than were observed in the normal surrounding tissue. tumors treated with paclitaxel-containing paste. The newly formed vessels entered the tumor from all angles, appearing as spokes attached to the center of a wheel (Figures 14A and 14B). Controlled tumors continued to increase in size and mass over the course of the

ES 2 207 451 T3 study. Hitologically, numerous dilated thin-walled capillaries were observed at the periphery of the tumor and few endothelial cells were observed to be in cell division. The tumor tissue was well vascularized and fully viable.

As an example, the following data was obtained on two tumors of similar size (initially, at the time of explantation) placed on the same CAM. For the tumor treated with thermopaste containing 20% ​​paclitaxel, the tumor measured 330 mm x 597 mm; the immediate periphery of the tumor had 14 blood vessels, while the tumor mass had only 3-4 small capillaries. For the tumor treated with thermopaste containing 20% ​​paclitaxel, the tumor measured 330 mm x 597 mm; the immediate periphery of the tumor had 14 blood vessels, while the tumor mass had only 3-4 small capillaries: for the thermopaste-treated tumor without content, the tumor size was 623 mm x 678 mm; the immediate periphery of the tumor had 14 blood vessels, while the tumor mass had only 34-4 small capillaries. For the thermopaste-treated tumor without content, the tumor size was 623 mm x 678 mm: the immediate periphery of the tumor had 54 blood vessels, while the tumor mass had 12-14 small blood vessels. Furthermore, the surrounding CAM itself contained many more blood vessels compared to the area surrounding the paclitaxel-treated tumor.

This study demonstrates that thermopaste releases sufficient amounts of paclitaxel to inhibit the pathological angiogenesis that accompanies tumor growth and development. Under these conditions, angiogenesis is maximally stimulated by tumor cells that produce angiogenic factors capable of inducing ingrowth of capillaries from the surrounding tissue into the tumor mass. Temopaste containing 20% ​​paclitaxel is capable of blocking this procedure and limiting the ability of tumor tissue to maintain an adequate blood supply. This results in a decrease in tumor mass through both a cytotoxic effect of the drug on the tumor cells themselves and by depriving the tissue of the nutrients required for growth and expansion.

Example 12

Inhibition of agiogenesis by paclitaxel

A. Chicken Chorioallantoic Membrane ("CAM") Assays

Fertilized chicken embryos were incubated for 3 days prior to shellless culture. In this procedure, the contents of the eggs were emptied by removing the shell placed around the air gap. The membrane inside the shells was then removed and the opposite end of the shell was pierced to allow the egg contents to slide smoothly from the blunt end. The egg contents were poured into sterilized round-bottom glass bowls and covered with petri dish lids. These were then placed in an incubator at a relative humidity of 90% and 3% CO2 and were incubated for 3 days.

Paclitaxel (Sigma, St. Louis, MI) was mixed at concentrations of 0.25, 0.5, 1, 5, 10, 30 µg per 10 µl aliquot of 0.5% aqueous mytylcellulose. Since paclitaxel is insoluble in water, glass granules were used to produce fine particles. 10 microliter aliquots of this solution were dried on paraffin for 1 hour to form 2 mm diameter discs. The dried paclitaxel-containing disks were then carefully placed on the growing edge of each CAM on day 6 of incubation. Controls were obtained by placing paclitaxel-free methylcellulose discs on the CAMs for the same length of time. After a 2-day exposure (incubation day 8), the vascularization was examined by means of a stereomicroscope. Liposyn II, a white opaque solution, was injected into the CAM to increase the visibility of vascular details. The vascularization of the live unstained embryos was imaged using a Zeiss stereomicroscope that was interfaced with a video camera (Dage-MTI Inc., Michigan City, IN). These video signals were then displayed at 160x magnification and captured using an image analysis system (Vidas, Contron; Etching, Germany). Image negatives were then prepared on a chart recorder (Model 3000; Matrix Instruments, Orangeburg, NY).

The membranes of the 8-day-old shellless embryos were flooded with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer; additional fixative was injected under the CAM. After 10 minutes in situ, the CAM was removed and placed in fresh fixative for 2 hours at room temperature. The tissue was then washed overnight in cadodilate buffer containing 6% sucrose. The areas of interest were subsequently fixed in 1% osmium tetroxide for 1.5 hours at 4 ° C. The tissues were then dehydrated in graduated series of ethanols, solvent exchanged with propylene oxide and embedded in Spurr resin. Thin sections were cut with a diamond blade, placed on copper grids, stained, and examined on a Joel 1200EX electron microscope. Similarly, 0.5 mm sections were cut and stained with toluene blue for light microscopy.

On day 11 of development, the chick embryos were used for the corrosion suppression technique. Mercox resin (Ted Pella, Inc., Redding, CA) was injected into the CAM vascularization using a 30 gauge hypodermic needle. The suppressor material consisted of 2.5 grams of Mercox CL-2B polymer and 0.05 grams of catalyst (55% benzoyl peroxide) having a polymerization time of 5 minutes. After injection, the plastic was allowed to settle in place for one hour at room temperature and then overnight in a

ES 2 207 451 T3 oven at 65 ° C. The CAM was then placed in a 50% aqueous sodium hydroxide solution to digest all the organic components. The plastic patches were extensively viewed in distilled water, air dried, gold / palladium coated, and viewed with the Philips 501B scanning electron microscope.

The results of the above experiments are shown in Figures 15-18. Briefly, the general characteristics of the normal chicken shell egg culture are shown in Figure 15A. On day 6 of incubation, the embryo is positioned centrally to a radially expanding network of blood vessels; CAM develops adjacent to the embryo. These growing vessels are located close to the surface and are easily visible making this system an idealized model for the study of angiogenesis. Live unstained capillary networks of the CAM can be imaged noninvasively with a stereomicroscope. Figure 15B illustrates such a vascular zone in which blood cell elements were recorded using a video / computer interface. The three-dimensional architecture of such CAM capillary networks is shown by the corrosion suppression method and was observed in the scanning electron microscope (Figure 15C). These deletions revealed underlying vessels projecting toward the CAM surface where they form a single layer of anastomotic capillaries.

Reverse sections through the CAM show an outer ectoderm consisting of a double cell layer, a broader mesodermal layer containing capillaries and underlying the ectoderm, adventitious cells, and a single endodermal inner layer (Figure 15D). At the electron microscope level, the typical structural details of CAM capillaries are demonstrated. Typically, these vessels are closely associated with the inner cell layer of the ectoderm (Figure 15E).

After 48 hours of exposure to paclitaxel at concentrations of 0.25, 0.5, 1, 5, 10 or 30 μβ, each CAM was examined under live conditions with a stereomicroscope equipped with a video / computer interface in order to evaluate the effects on angiogenesis. This constitution of the images was used at a magnification of 160 times that allowed the direct visualization of the blood cells in the capillaries; thus the blood flow in the areas of interest could be easily assessed and recorded. For this study, inhibition of angiogenesis was defined as an area of ​​the CAM (measuring 2-6 mm in diameter) that lacks a capillary network and vascular blood flow. In all experiments, the avascular zones were assessed on a 4-point avascular gradient (Table 1). This scale represents the degree of global inhibition, the maximum inhibition being represented as a 3 on the avascular gradient scale. Paclitaxel was highly congruent and induced a maximum evascular zone (6 mm in diameter or a 3 on the avascular gradient scale) within 48 hours, depending on its concentration.

TABLE 1

Avascular gradient

- - normal vascularization

- - lack of any microvascular movement

2 * - - small avascular area approximately 2 mm in diameter 3 * - - avascularization extending beyond the disc (6 mm in diameter) * - indicates a positive antiangiogenesis response

Dose-dependent experimental data of the effects of various therapeutic agents at different concentrations are shown in Table 2.

TABLE 2

Paclitaxel agent

Supply vehicle

Concentration Xnhibition / n

<td>methylcellulose (1C</td><td>1 μΐ)</td><td>0.25 μα</td><td> 2/11</td>

<td>methylcellulose (1C</td><td>• μΐ)</td><td>0.5 μα</td><td> 6/11</td>

<td>methylcellulose (1C</td><td>»Μΐ)</td><td>1 μ &amp;</td><td> 6/15</td>

<td>methylcellulose (1C</td><td>1 μΐ)</td><td>5 ug</td><td> 20/27</td>

<td>methylcellulose (1C</td><td>1 μΐ)</td><td>ίο μα</td><td> 16/21</td>

<td>methylcellulose (1C</td><td>1 μΐ)</td><td>30 μα</td><td> 31/31</td>

<td>PCL paste (3</td><td>mg)</td><td> 0,05%</td><td> 0/9</td>

<td>PCL paste (3</td><td>mg)</td><td> 0,10%</td><td> 1/8</td>

<td>PCL paste (3</td><td>mg)</td><td> 0,25%</td><td> 5/7</td>

<td>PCL paste (3</td><td>mg)</td><td> 0,5%</td><td> 4/4</td>

<td>PCL paste (3</td><td>mg)</td><td> 1%</td><td> 8/8</td>

<td>PCL paste (3</td><td>mg)</td><td> 2%</td><td> 10/10</td>

<td>PCL paste (3</td><td>mg)</td><td> 5%</td><td> 10/10</td>

<td>PCL paste (3</td><td>mg)</td><td> 10%</td><td> 9/9</td>

<td>PCL paste (3</td><td>mg)</td><td> 20%</td><td> 6/6</td>

ES 2 207 451 T3

<td>Agent</td><td>Supply vehicle</td><td>Concentration traction</td><td>Inhibition / n</td>

<td></td><td>20% gelatin: 60% PCL paste {3 mg)</td><td> 20%</td><td> 5/6</td>

<td></td><td>gelatin (1 mg)</td><td> 20%</td><td> 17/17</td>

<td></td><td>ophthalmic suspension (2x10 pl)</td><td> 0,3%</td><td> 1/12</td>

<td></td><td>ophthalmic suspension (2x15 μΐ)</td><td> 0,3%</td><td> 3/3</td>

<td></td><td>ophthalmic suspension (1x15 μΐ)</td><td> 0, 3%</td><td> 15/15</td>

<td></td><td>ophthalmic microsphere suspension (15 μΐ)</td><td> 10%</td><td> 4/4</td>

<td></td><td>stent coating (~ 1 mg)</td><td> 2, 5%</td><td> 5/5</td>

<td></td><td>stent lining (- 1 mg)</td><td> 10%</td><td> 1/1</td>

<td></td><td>stent lining (- 1 mg)</td><td> 33%</td><td> 3/3</td>

<td></td><td>cyclodextrin solution (10 μΐ)</td><td> 10%</td><td> 5/5</td>

<td></td><td>dry micellar formulation (1 mg)</td><td> 10%</td><td>too toxic</td>

<td></td><td>micellar solution (10 μΐ)</td><td> 10%</td><td>too toxic</td>

<td></td><td>micellar solution (10 μΐ)</td><td>4 pg</td><td>1/1 - too toxic</td>

<td></td><td>Cremophor Taxol (10 μΐ)</td><td>4 pg</td><td>1/1 - too toxic</td>

<td></td><td>4PCL flakes: lMePEG (1mg)</td><td> 20%</td><td> 10/13</td>

<td></td><td>PCL paste: MePEG (3 mg)</td><td> 20%</td><td> 6/9</td>

<td></td><td>microspheres (mucoadhesive)</td><td> 20%</td><td> 7/7</td>

<td></td><td>microspheres (EVA)</td><td> 0,6%</td><td> 2/2</td>

<td></td><td>microspheres (30-10 pm) -slow release</td><td> 20%</td><td> 11/11</td>

<td></td><td>microspheres (30-10 pm) -slow release</td><td> 10%</td><td> 1/8</td>

<td></td><td>microspheres (30-10 pm) - medium release</td><td> 20%</td><td> 5/6</td>

<td></td><td>microspheres (30-10 pm) - medium release</td><td> 10%</td><td> 5/9</td>

<td></td><td>microspheres (30-10 pm) - rapid release</td><td> 20%</td><td> 8/11</td>

<td></td><td>microspheres (30-10 pm) - rapid release</td><td> 10%</td><td> 9/9</td>

<td>bacatina</td><td>paste (2 mg)</td><td>2 pg</td><td> 2/3</td>

<td></td><td>methylcellulose (5 pl)</td><td>5 pg</td><td> 4/7</td>

<td>methotrexate</td><td>PCL paste (3 mg)</td><td> 1%</td><td> 0/13</td>

<td></td><td>PCL paste (3 mg)</td><td> 2%</td><td> 0/3</td>

<td></td><td>PCL paste (3 mg)</td><td> 20%</td><td> 0/1</td>

<td></td><td>PCL paste: MePEG (3 mg)</td><td> 2%</td><td> 1/1</td>

<td></td><td>95PCL paste: 5MePEG (3mg)</td><td> 1%</td><td> 0/6</td>

<td></td><td>95PCL paste: 5MePEG (3mg)</td><td> 10%</td><td> 0/5</td>

<td></td><td>methylcellulose (10 pl)</td><td>2 pg</td><td> 0/8</td>

<td>acetate</td><td>ophthalmic suspension (2x10 pl)</td><td> 1%</td><td> 3/4</td>

<td colspan="4">prednisolone</td>

<td></td><td>ophthalmic suspension (2x10 pl)</td><td> 1%</td><td> 1/1</td>

<td>pynogenol</td><td>methylcellulose (10 pl)</td><td>10 pg</td><td> 1/18</td>

<td colspan="4">(proanthocy- nidine)</td>

<td></td><td>PCL paste (3 mg)</td><td> 15%</td><td> 1/2</td>

<td></td><td>PCL paste (3 mg)</td><td> 30%</td><td>too toxic</td>

<td>verotoxin</td><td>methylcellulose (10 pl)</td><td>10 ng</td><td> 0/8</td>

<td></td><td>methylcellulose (10 pl)</td><td>675 ng</td><td> 0/2</td>

ES 2 207 451 T3

<td>Agent</td><td>Supply vehicle</td><td>Concentration traction</td><td>Inhibition / n</td>

<td>heparan sulfate</td><td>methylcellulose (10 μΐ)</td><td>0.1 pg</td><td> 0/6</td>

<td colspan="4">fragment (1)</td>

<td>heparan sulfate</td><td>methylcellulose (10 μΐ)</td><td>0.4 pg</td><td> 0/7</td>

<td colspan="4">fragment (2)</td>

<td>vanadate</td><td>microspheres (1 mg)</td><td> 5%</td><td> 0/5</td>

<td>vanadyl sulfate</td><td>PCL paste (3 mg)</td><td> 2,5%</td><td> 0/3</td>

<td>BMOV</td><td>PCL paste (3 mg)</td><td> 10%</td><td>too toxic</td>

<td></td><td>PCL paste (3 mg)</td><td> 25%</td><td>too toxic</td>

<td></td><td>PCL paste (3 mg)</td><td> 35%</td><td>too toxic</td>

<td>BEOV</td><td>PCL paste (3 mg)</td><td> 10%</td><td>too toxic</td>

<td>s-phosphonate</td><td>80% PLA paste: 20% MePEG (1mg) PCL paste (1 mg) PCL paste (3 mg) PCL paste (3 mg) PCL paste (3 mg) PCL paste (3 mg)</td><td> 20% 2% 1% 2% 4% 8%</td><td>too toxic 2/7 0/9 0/6 0/3 1/9</td>

<td>tamoxifen cartilage powder</td><td>methylcellulose (10 μΐ) N / A</td><td>5 pg 1 mg</td><td> 0/2 0/5</td>

<td colspan="4">shark</td>

<td>estramustine sodium</td><td>PCL paste (3 mg)</td><td> 5%</td><td> 0/6</td>

<td>phosphate</td><td>PCL paste (3 mg)</td><td> 10%</td><td> 0/6</td>

<td></td><td>methylcellulose (10 μΐ)</td><td>9 pg</td><td>too toxic</td>

<td>vinblastine</td><td>methylcellulose (10 μΐ) PCL paste (3 mg) PCL paste (3 mg) PCL paste (3 mg)</td><td>2 pg 0.25% 0.5% 1%</td><td>too toxic 4/6 0/4 2/3</td>

<td></td><td>PCL paste (3 mg)</td><td> 2%</td><td>too toxic</td>

<td>vincristine</td><td>methylcellulose (10 μΐ)</td><td>9 pg</td><td>too toxic</td>

<td></td><td>methylcellulose (10 μΐ)</td><td>i ug</td><td>too toxic</td>

<td></td><td>PCL paste (3 mg)</td><td> 0,05%</td><td>1/1 - too toxic</td>

<td></td><td>PCL paste (3 mg)</td><td> 0,1%</td><td>2/2 - too toxic</td>

<td></td><td>PCL paste (3 mg)</td><td> 0,25%</td><td>1/1 - too toxic</td>

<td></td><td>PCL paste (3 mg)</td><td> 0,5%</td><td>too toxic</td>

<td></td><td>PCL paste (3 mg)</td><td> 1%</td><td>too toxic</td>

<td></td><td>PCL paste (3 mg)</td><td> 2%</td><td>too toxic</td>

<td>diterpene-1</td><td>methylcellulose (10 μΐ)</td><td>3 pg</td><td> 0/5</td>

<td>diterpene-2</td><td>methylcellulose (10 μΐ)</td><td>3 pg</td><td> 0/5</td>

<td>lavendustine-c</td><td>PCL paste (3 mg) PCL paste (3 mg)</td><td> 10% 20%</td><td> 0/14 0/10</td>

<td>MDHC (inhibitor of</td><td>PCL paste (3 mg)</td><td> 20%</td><td> 0/8</td>

<td colspan="4">tyrosine)</td>

<td>erbstatin</td><td>PCL paste (3 mg)</td><td> 20%</td><td>0/5 - too toxic</td>

<td>genistein</td><td>PCL paste (3 mg)</td><td> 10%</td><td> 0/7</td>

ES 2 207 451 T3

<td>Agent</td><td>Supply vehicle</td><td>Concentration</td><td>Inhibition / n</td>

<td></td><td>PCL paste (3 mg)</td><td> 20%</td><td> 0/4</td>

<td>herbimysin</td><td>PCL paste (3 mg)</td><td> 2%</td><td> 3/4</td>

<td></td><td>PCL paste (3 mg)</td><td> 0,5%</td><td> 1/1</td>

<td>captothecin</td><td>PCL paste (3 mg)</td><td> 0,25%</td><td> 3/4</td>

<td></td><td>PCL paste (3 mg)</td><td> 1%</td><td> 2/3</td>

<td></td><td>PCL paste (3 mg)</td><td> 5%</td><td> 4/5</td>

<td></td><td>methylcellulose (10 μϊ)</td><td>20 μα / 70 μg</td><td> 2/4</td>

<td>Suramine and acetate</td><td>methylcellulose (10 μϊ)</td><td>50 μα / 40 μg</td><td> 5/14</td>

<td>cortisone</td><td>methylcellulose (10 μϊ)</td><td>50 μg / 50 μg</td><td> 3/26</td>

<td></td><td>methylcellulose (10 μϊ)</td><td>20 μg / 50 μg</td><td> 0/24</td>

<td>Sumarin and sulfate</td><td>methylcellulose (10 μϊ)</td><td>70 μg / 70 μα</td><td> 0/9</td>

<td>tetrahydro-S-protamine</td><td>methylcellulose (10 μΐ)</td><td>50 μα / 50 μα</td><td> 0/6</td>

<td></td><td>methylcellulose (10 μΐ)</td><td>50 μα</td><td> 0/10</td>

<td></td><td>methylcellulose (10 μΐ)</td><td>100 μα</td><td> 1/10</td>

<td>1 JLMr</td><td>methylcellulose (10 μΐ)</td><td>15 μα</td><td> 0/5</td>

<td>colchicine</td><td>methylcellulose (10 μϊ)</td><td>3 μα</td><td>1/1 - dema-</td>

<td></td><td></td><td></td><td>too toxic</td>

Typical paclitaxel-treated CAMs are also shown in the clear methylcellulose disk positioned centrally over the avascular zone, measuring 6mm in diameter. At slightly higher magnification, the periphery of such avascular areas is clearly apparent (Figure 16C); the surrounding functional vessels were often redirected outward from the source of paclitaxel (Figures 16C and 16D). Such angular redirection of blood flow was never observed under normal conditions. Another characteristic of the effects of paclitaxel was the formation of blood islands in the avascular zone, representing the aggregation of blood cells.

The associated morphological alterations of CAM treated with paclitaxel are already readily apparent at both light and electron microscope levels. For the convenience of presentation, three distinct phases of the general transition from normal to avascular are shown. Near the periphery of the avascular zone, the CAM is highlighted by an abundance of mitotic cells within all three layers of germs (Figures 17A and 18A). This increased mitotic division was also a consistent observation for capillary endothelial cells. However, endothelial cells remained intact in splicing without extravasation of blood cells. With further degradation, CAM is characterized by capillary breakage and dissolution (Figures 17B and 18B). Presumptive endothelial cells, typically arrested in mitosis, still maintain a close spatial relationship with blood cells and lie underlying the ectoderm; however, these cells are not spliced. The most central part of the avascular zone was characterized by a thickened ectodermal and endodermal layer (Figures 17C and 18C). Although these layers were thickened, the cell splices remained intact and the layers maintained their structural characteristics. Within the mesoderm, mitotically detected scattered cells were abundant; these cells did not exhibit the endothelial cell polarization observed in the first phase. Also, throughout this avascular zone, degenerating cells were common as seen by dense electron vacuoles and cellular debris (Figure 18C).

In summary, this study demonstrated that 48 hours after the application of paclitaxel to the CAM, angiogenesis was inhibited. The inhibition of blood vessels formed as an avascular zone that was represented by the three transition phases of the effect of paclitaxel. The most affected central area of ​​the avascular area contained interrupted capillaries with extravasated red blood cells; this indicated that intracellular junctions between endothelial cells were absent. The cells of the endoderm and the ectoderm maintained their intercellular junctions and, therefore, these layers of germs remained intact; however, they were slightly thickened. As they approached the normal avascular zone, the blood vessels retained their junction complexes and therefore also remained intact. At the periphery of the paclitaxel-treated area, vessel growth was further inhibited, which was evident by the typical redirection or "kinking" effect of blood vessels (Figure 16D).

Paclitaxel-treated avascular areas also revealed an abundance of cells arrested in mitosis in all three germ layers of the CAM; this was unique to paclitaxel as no previous study had illustrated such an event. Being arrested in mitosis, endothelial cells were unable to undergo their normal metabolic functions involved in angiogenesis. In comparison, the avascular zone formed by suramin and cortisone acetate did not produce mitotically arrested cells in CAM; they only prevented the growth of additional blood vessels in the treated area. Therefore, even though these agents are anti-angiogenic, there are many points where the angiogenesis procedure can be targeted.

The effects of paclitaxel were also observed during the 48 hour period. During this observation period, angiogenesis inhibition was seen to occur early, 9 hours later on application. Histological sections revealed a similar morphology as observed in the first transition phase of the avascular zone at 48 hours, illustrated in Figures 17A and 18A. It was also observed in the revascularization procedure in the previously observed avascular area. It has been found that the avascular zone formed by heparin and angiostatic steroids was revascularized 60 hours after application. In one study,

ES 2 207 451 T3 avascular areas treated with paclitaxel did not revascularize for at least 7 days after application, which implies a more potent long-term effect.

Example 13

Effect of paclitaxel and campothecin on LNCaP cell proliferation

Materials and methods

LNCaP cells were seeded at concentrations of 2x10³ and 1x10³ cells / well, respectively, in 96-well plates. After 48 hours, different concentrations of paclitaxel or campothecin (25 μθ in each culture well were added and the plates were incubated at 37 ° C for 5 days. After incubation, the cells were terminated with 1% gluteraldehyde solution , and were stained for 5 minutes with 0.5% crystal violet. The dye was successively eluted with 100 µl of buffer solution and the absorbance was read on a Titertek Multiskan microplate reader using an absorbance wavelength of 492 mm. Cell growth was expressed as a percentage relative to control wells in the absence of compound (adjusted to 100%). Results

Paclitaxel inhibited the growth of LNCaP cells with an EC50 of approximately 0.09 nM. Apoptosis experiments were performed on the cells in the wells after treatment with paclitaxel using DNA fragmentation assays. Extensive cell apoptosis was observed indicating that paclitaxel was cytotoxic via an apoptotic mechanism.

Campothecin was extremely potent in its cytotoxic action against LNCaP cells. Concentrations as low as 0.001 nM were toxic to over 60% of the cells. Therefore, the EC50 for this drug against LNCaP cells should be in the range of femtomolar concentrations.

TABLE 1

<td>N</td><td>Paclitaxel I (nM)</td><td>Absorbance 492 nm</td><td>% Increase</td>

<td> 16</td><td> 0,001</td><td> 0,049±0,05</td><td> 100</td>

<td> 16</td><td> 0,01</td><td> 0,04±0,03</td><td> 81</td>

<td> 8</td><td> 0,05</td><td> 0,36±0,02</td><td> 73</td>

<td> 8</td><td> 0,1</td><td> 0,20±0,03</td><td> 40</td>

<td> 8</td><td> 1</td><td> 0,025±0,01</td><td> 5</td>

<td> 8</td><td> 10</td><td> 0,027±0,01</td><td> 5</td>

<td> 8</td><td> 100</td><td> 0,033±0,01</td><td> 6</td>

Control 492 nm absorbance = 0.49 ± 0.06

TABLE 2

<td>N</td><td>Paclitaxel I (nM)</td><td>Absorbance 492 nm</td><td>% Increase</td>

<td> 16</td><td> 0,001</td><td> 0,169±0,05</td><td> 36</td>

<td> 8</td><td> 0,05</td><td> 0,14±0,04</td><td> 29</td>

<td> 8</td><td> 0,1</td><td> 0,10±0,02</td><td> 21</td>

<td> 8</td><td> 1</td><td> 0,10±0,02</td><td> 21</td>

<td> 8</td><td> 10</td><td> 0,088±0,02</td><td> 17</td>

<td> 15</td><td> 100</td><td> 0,038±0,01</td><td> 8</td>

Control 492 nm absorbance = 0.47 ± 0.05

Example 14

Anti-angiogenesis activity of additional anti-microtubule agents

In addition to paclitaxel, other anti-microtubule agents can be incorporated into polymeric carriers. Representative examples provided below include comptothecin and vinca alkaloids such as vinblastine and vincristine, and microtubule stabilizing agents such as tubercidin, aluminum fluoride, and LY290181.

ES 2 207 451 T3

A. Incorporation of PCL agents

The agents were crushed with a hammer mortar to reduce the particle size to below 5 microns. These were then mixed in the form of a dry powder with polycaprolactone (molecular weight 18,000, Birmingham Polymers. AL USA). The mixture was heated at 65 ° C for 5 minutes and then the polymer / agent melt mixture was stirred into a smooth paste for 5 minutes. The melt was then collected in a 1 ml syringe and extruded to form 3 mg granules. These granules are then placed on the CAM on day 6 of gestation to assess their anti-angiogenic properties.

B. Effects of campothecin-containing PCL paste on CAM

The campothecin-containing thermopaste was effective in inhibiting angiogenesis when compared to the PCL control granules. For a drug content of 5%, 4/5 of the CAMs tested showed potent inhibition of angiogenesis. Furthermore, for contents of 1% and 0.25%, 2/3 and 3/4 of the CAMs showed inhibition of angiogenesis, respectively. Therefore, it is evident from these results that campothecin was sufficiently released from PCL thermopaste and has anti-angiogenic therapeutic efficacy.

C. Effects of PCL paste containing vinblastine and vincristine on CAM

When the formulations were tested on CAM, it was apparent that the agents being released from the PCL granule in amounts sufficient to induce a biological effect. Both vinblastine and vincristine induced anti-angiogenic effects in the CAM assay when compared to control PCL thermopaste granules.

At concentrations of 0.5% and 0.1% drug content, vincristine induced an inhibition of angiogenesis in all CAMs tested. When concentrations exceeded 2% were tested, toxic drug levels were achieved and unexpected embryonic death occurred.

Vinblastine was also effective in inhibiting angiogenesis in CAM at concentrations of 0.25%, 0.5%, and 1%. However, at concentrations exceeding 2%, vinblastine was toxic to the embryo.

D. Effects of PCL paste with tubercidin content on CAM

The tubercidin-containing paste was effective in inhibiting angiogenesis when compared to control granules. For a drug content of 1%, tubercidin induced an angiogenesis inhibition in 1/3 of the CAMs tested. However, at higher drug concentrations of 5% drug content, tubercidin potently inhibited angiogenesis in 2/3 of CAMs. Therefore, it was evident from these results that tubercidin was sufficiently released from the PLC paste and that it has potent anti-angiogenic activity.

E. Effects of PCL paste containing aluminum fluoride on CAM

PCL pastes containing aluminum fluoride (AlF3) were effective in inhibiting angiogenesis for a drug content of 20% compared to control granules. For a drug content of 20%, 2/4 of the CAMs showed inhibition of angiogenesis as evidenced by an avascular zone measuring between 2 and 6 mm in diameter. However, for a lower drug content, 1% and 5%, inhibition of angiogenesis was not evident (0/6 and 0/5 CAMs, respectively). Therefore, aluminum fluoride was effective in inducing angiogenesis inhibition only at higher drug concentrations.

F. Effect of PCL paste containing LY290181 on CAM

Titration of the PCL paste containing 5% LY290181 on the CAM showed that LY290818 induced an angiogenesis inhibition in 1/3 of the CAMs tested. However, for a drug content of 1%, LY290181 did not induce any anti-angiogenesis response (n = 2).

Example 15

Effect of paclitaxel on the viability of non-proliferating cells

Although it is important that a disease modifying agent is able to strongly inhibit a variety of cellular activities (proliferation, inflammation, production of proteolytic enzymes) that are produced in excess during the development of chronic inflammation, it must not be toxic to normal tissues. . It is particularly critical that normal cells do not deteriorate, as this would lead to disease progression. In this example, the effect of paclitaxel on the viability of normal non-dividing cells was examined using cultured chondrocytes grown to confluence.

Briefly, the chondrocytes were incubated in the presence (10^-5 M, 10^-7 M and 10^-9 M) or absence (control) of paclitaxel for 72 hours. At the end of this time period, the total number of viable cells was visually determined by trypan blue dye exclusion. This experiment was performed 4 times and the data was contrasted.

ES 2 207 451 T3

The results of this experiment are shown in Figure 21. Briefly, as evident from Figure 21, paclitaxel does not affect the viability of normal non-proliferating cells in vitro even at high concentrations (10^-5 M) of paclitaxel. More specifically, even at drug concentrations sufficient to block the pathological procedures described in the preceding examples, there was no cytotoxicity to normal chondrocytes. Example 16

Selection of penetration enhancer for a topical formulation of paclitaxel

A. Solubility of paclitaxel in various enhancers

The following penetration enhancers were examined: Transcutol^®, ethanol, propylene glycol, isopropyl myristate, oleic acid and Transcutol: isopropyl myristate (9: 1 v: v). One milliliter of each improver in glass vials was preheated to 37 ° C and excess paclitaxel was added. A 0.5 ml sample of the fluid from each vial was centrifuged at 37 ° C and 13000 rpm for 2 minutes. Aliquots (0.1 ml) of supernatants from the centrifuge tubes were transferred to volumetric flasks and diluted with methanol. The content of paclitaxel was assessed by high pressure liquid chromatography (HPLC).

B. Partition coefficient

A specific amount of paclitaxel was dissolved in a volume of builder heated to 37 ° C. Aliquots (1 ml) of this solution were added to 1 ml of octanol in a 4 ml glass vial. Phosphate buffered saline (1 ml) (pH 7.4) was then added and the vials were centrifuged to create an emulsion. The vials were placed in an oven at 37 ° C for 16 hours, after which 0.1 ml of the octanol phase was removed from each vial and diluted with 9.9 ml of methanol. For the aqueous phases, 0.5 ml samples were taken from the oleic acid and isopropyl myristate vials and 0.5 ml samples were taken from the propylene glycol vials and diluted with 0.5 ml methanol. From the Transcutol vials, 0.1 ml samples were taken and diluted with 9.9 ml of Transcutol: PBS 50:50 and 0.1 ml samples were taken from the ethanol vials and diluted with 50:50 ethanol: PBS. The content of paclitaxel was determined by HPLS. Each determination was made in triplicate.

C. Results

TABLE 1

Saturation Paclitaxel Concentration in Various Penetration Enhancers

<td></td><td>Concentration of</td><td>paclitaxel (mg / ml)</td>

<td>Improver</td><td>Half</td><td>Typical deviation</td>

<td>Transcutol®</td><td> 346,85</td><td> 2,59</td>

<td>Ethanol</td><td> 68,91</td><td> 3,49</td>

<td>Propylene glycol</td><td> 21,56</td><td> 0,11</td>

<td>Isopropyl myristate</td><td> 0,43</td><td> 0,01</td>

<td>Oleic acid</td><td> 0,31</td><td> 0,01</td>

<td>Transcutol®: isopripyl myristate (9: 1 v: v)</td><td> 353,93</td><td> 0,42</td>

The octanol / water partition coefficients, Ko / w, are shown in Table 2.

TABLE 2

Octanol / water partition coefficient of paclitaxel in various builder solutions

<td>Improver</td><td>^Ko / w</td><td>Typical deviation</td>

<td>Transcutol®</td><td> 25,25</td><td> 0,27</td>

<td>Ethanol</td><td> 6,88</td><td> 0,13</td>

<td>Propylene glycol</td><td> 37,13</td><td> 2,48</td>

<td>Isopropyl myristate</td><td>TO</td><td> -</td>

<td>Oleic acid</td><td>TO</td><td> -</td>

ES 2 207 451 T3

To work effectively, paclitaxel must penetrate the skin to the lower layers of the viable epidermis. It has been established that for drugs to penetrate the epidermis reliably, they must have an octanol / water partition coefficient of or close to 100 (Hadgraft JH and Walters K., Drug absorption enhancements, AG de Boers Ed., Harwood Publishers , 1994). Based on the results of Tables 1 and 2, Propylene Glycol and Transcutol show the best combination of solubilization of paclitaxel and the increase in its partition of an oil phase relative to an aqueous phase.

However, the Ko / w produced by both Transcutol and propylene glycol can be somewhat low, therefore they were combined with isopropyl myristate which has infinite Ko / w in an attempt to increase the solubility of paclitaxel in the octanol phase . Isopropyl myristate and Transcutol were mixed in a 1: 9 volume ratio. The isopropyl myristate dissolved easily at room temperature in the Transcutol. In order to form a homogeneous phase, propylene glycol and isopropyl myristate were also mixed with ethanol in a 4: 3.5: 0.5 ratio of propylene glycol: ethanol: isopropyl myristate. The Ko / w results are shown in Table 3.

TABLE 3

Octanol / water partition coefficients of paclitaxel in improver

Combinations

<td>Improver</td><td>^Ko / w</td><td>Typical deviation</td>

<td>Transcutol® isopropyl myristate (9: 1)</td><td> 43,45</td><td> 0,43</td>

<td>Propylene Glycol: Ethanol: Isopropyl Myristate (4.0: 3.5: 0.5)</td><td> 42,39</td><td> 1,66</td>

The addition of isopropyl myristate to Transcutol resulted in a significant increase in the partition coefficient. However, the propylene glycol: ethanol: isopropyl myristate solution did not result in a significant improvement in partition coefficient over that of propylene glycol alone. This latter result, and the fact that ethanol had been found to exacerbate the psoriatic state, effectively removed this enhancing combination from further consideration. Furthermore, the addition of isopropyl myristate actually increased the solubility of paclitaxel over its solubility in Transcutol alone. The solubility of paclitaxel in Transcutol was 346.9 mg / ml, while in a combination of Transcutol: isopropyl myristate, the solubility increased to 353.9 mg / ml. Therefore, this enhancer combination was chosen in the skin studies.

Example 17

Preparation and Analysis of Topical Formulations of Paclitaxel

A. Preparation of Paclitaxel Ointment A

Transcutol (3.2 g), isopropyl myristate (0.3 g), labrasol (3.5 g), paclitaxel (0.01 g) and 0.5 mCi / ml H were combined.³-paclitaxel (0.3 ml) in a 20 ml scintillation vial. In a separate scintillation vial, labrafil (2.5 g), arlacel 165 (1.2 g) and compritol (0.3 g) were combined and heated to 70 ° C until completely melted. The contents of the first scintillation vial were added to the melt, centrifuged until homogeneous, and allowed to cool.

B. Preparation of Paclitaxel Ointment B

Transcutol (2.5 g), isopropyl myristate (1.0 g), labrasol (2.5 g), paclitaxel (0.01 g) and 0.5 mCi / ml H were combined³-paclitaxel (0.3 ml) in a 20 ml scintillation vial. In a separate scintillation vial, labrafil (2.5 g), arlacel 165 (1.2 g) and compritol (0.3 g) were combined and heated to 70 ° C until completely melted. The contents of the first scintillation vial were added to the melt, centrifuged until homogeneous, and allowed to cool.

C. Skin preparation and penetration study

Cut and frozen Yucatán mini guinea pig skin was stored at -70 ° C until used. Skin samples were prepared using a # 10 cork piercer to pierce discs from the frozen skin. The samples were rinsed with a streptomycin-penecillin solution and placed in freezer bags and stored at -70 ° C.

Skin sections were placed in Franz diffusion cells, with the stratum corneum facing up. The lower receptor solution was a 0.5% amoxicillin solution in water. A donor cell was retained on each surface of the skin. The paclitaxel ointment was heated until melted (40 to 50 ° C) and was withdrawn into a syringe. While

ES 2 207 451 T3 was still molten, 0.1 ml was extruded on each skin surface. The donor cells were covered with a glass disk and the whole was left for 24 hours.

After 24 hours, the cells were disassembled, the excess ointment was removed and stored in a scintillation vial. The skin surface was quickly washed with 3 ml of dichloromethane (DCM) and dried. The DCM was stored in the same vial as the excess ointment. The skin sections and receptor solution were placed in separate scintillation vials. The skin was cryotomized at -30 ° C in 30 μιη sections and collected in separate glass vials. The cut skin samples were dissolved by adding 0.5 ml of tissue solubilizer to each vial. The samples were left overnight to dissolve at room temperature. The next day, 3 ml of scintillation pool was added to the vials. For the DCM wash solutions, 100 µl was transferred to 1 ml of acetonitrile and then 3 ml of scintillation pool was added. The radioactivity of all solutions was measured using a beta counter.

Skin samples were placed in the Franz diffusion cells and separated into three groups. Each sample was treated accordingly (no treatment or ointment B with or without paclitaxel). After 34 hours, the samples were removed and treated using standard histological techniques.

C. Results

From the histological sections, the untreated skin stratum corneum section was found to be between 50 and 120 µm thick, while the viable epidermis was between 400 and 700 µg thick. For the ointment containing 3% w / w isopropyl myristate (ointment A), the concentration of paclitaxel in the skin was essentially constant at 1 μg / ml (1.2 x 10^-6 M) in the stratum corneum and throughout the viable epidermis. For the ointment containing 10% w / w isopropyl myristate (ointment B), the concentration of paclitaxel was constant in the stratum corneum and viable epidermis, but higher in the stratum corneum (6 μg / ml vs. 2 μg / ml). There was no radioactivity in the receptor solution for each ointment investigation, indicating that the paclitaxel did not pass completely through the skin section.

No major differences were seen when the ointment containing paclitaxel was applied.

Example 18 (Reference example)

Development of systemic formulations of paclitaxel for the treatment of psoriasis

In severe cases of psoriasis, the more aggressive treatments are considered acceptable and, therefore, the toxicities associated with systemic treatment with paclitaxel may be acceptable.

The systemic formulation for paclitaxel is comprised of amphiphilic diblock copolymers that, in aqueous solutions, mecile forms consisting of a hydrophobic core and a hydrophilic shell in water. Diblock copolymers of poly (DL-lactide) -blockmethoxypolyethylene glycol (PDLLA-MePEG), polycaprolactone-block-methoxy-polyethylene glycol (PCL-MePEG) and poly- (DL-lactide-cocaprolactone) -block-methoxy-polyethylene glycol (PDLLACLACL) can be synthesized. -MePEG) using a mass melt polymerization procedure, or similar methods. Briefly, given amounts of DL-lactide, caprolactone, and methoxypolyethylene glycols monomers with different molecular weights were heated (130 ° C) to melting under nitrogen bubbling and stirred. The stannous octoate catalyst (0.2% w / w) was added to the molten monomers. The polymerization was carried out for 4 hours. Molecular weights, critical micelle concentrations, and maximum paclitaxel contents were measured with GPC, fluorescence, and solubilization assays, respectively (Figure 22). High transport capacities of paclitaxel were obtained. The ability to solubilize paclitaxel depends on the compositions and concentrations of the copolymers (Figures 22 and 23). PDLLA-MePEG provided the most stable solubilized paclitaxel (Figures 23 and 24).

The strong association in the inner core of the polymeric micelles presents an environment of high capacity to transport hydrophobic drugs such as paclitaxel. Drugs can be covalently coupled to block copolymers to form a micellar structure or they can be physically incorporated into the hydrophobic cores of the micelles. Mechanisms of drug release from micelles include diffusion from the nucleus and exchange between single chain polymers and micelles. The small size of the micelles (typically less than 100 nm) will eliminate the difficulties associated with the injection of larger particles.

Example 19

Procedure for producing thermopaste

Five grams of polycaprolactone of molecular weight 10,000 to 20,000; (Polyscienes, Warrington Penn. USA) A 20 ml glass scintillation vial was placed in a 600 ml beaker containing 50 ml of heavy water. The beaker was gently heated to 65 ° C and held at that temperature for 20 minutes until the polymer melted. A known weight of paclitaxel, or other angiogenesis inhibitor, was thoroughly mixed into the molten polymer at 65 ° C. The molten polymer was poured into a previously heated mold (60 ° C, oven) and allowed to cool until the polymer

ES 2 207 451 T3 solidified. The polymer was cut into small pieces (approximately 2mm by 2mm in size) and placed in a 1ml glass syringe.

The glass syringe was then placed upside down (capped end down) in a 500 ml glass beaker containing 65 ° C distilled water (corning hot plate) until the polymer was completely melted. The dip was then inserted into the syringe to compress the molten polymer into a sticky mass at the tip end of the barrel. The syringe was capped and allowed to cool to room temperature.

For application, the syringe was again heated to 60 ° C and administered in the form of a liquid that solidified when cooled to room temperature.

Example 20

Modification of Paclitaxel Release from Thermopaste Using PDLLA-PEG-PDLLA and Low Molecular Weight Poly (D, L-Lactic Acid

A. Preparation of PDLLA-PEG-PDLLA and low molecular weight DPLLA

DL-lactide was purchased from Aldrich. Polyethylene glycol (PEG) with a molecular weight of 8,000, stannous octoate and DL-lactic acid were obtained from Sigma. Poly ^ -caprolactone (PCL) with a molecular weight of 20,000 was obtained from Birmingham Polymers (Birmingham, AL). Paclitaxel was purchased from Hauser Chemicals (Boulder, CO). Polystyrene standards with narrow molecular weight distributions were purchased from Polysciences (Warrington, PA). Acetonitrile and methylene chloride were HPLC grade (Fischer Scientific).

The PDLLA-PEG-PDLLA triblock copolymer was synthesized by ring-opening polymerization. DL-lactide and PEG monomers in different ratios were mixed and 0.5% by weight of 0.5% by weight stannous octoate was added. The polymerization was carried out at 150 ° C for 3.5 hours. Low molecular weight PDLLA was synthesized by means of DL-lactic acid polycondensation. The reaction was carried out in a glass flask under the conditions of gentle nitrogen purge, mechanical stirring and heating at 180 ° C for 1.5 hours. The molecular weight of PDLLA was approximately 800, measured by titrating the carboxylic acid end groups.

B. Preparation of pasta formulations

Paclitaxel with contents of 20% or 30% was thoroughly mixed into PDLL-APEG-PDLLA copolymers or PDLLA: PCL 90:10, 80:20 and 70:30 blends melted at approximately 60 ° C. The paclitaxel-containing pastes were weighed into 1 ml syringes and stored at 4 ° C.

C. Characterization of PDLLA-PEG-PDLLA and the paste mixtures

The molecular weights and distributions of the PDLLA-PEG-PDLLA copolymers were determined at room temperature by GPC using a Shimadzul LC-10AD HPLC pump and a RID6A Shimadzu refractive index detector (Kyoto, Japan) coupled to a gel column. Hewlett Packard P1 of 10⁴. The mobile phase was chloroform with a flow rate of 1 ml / minute. The injection volume of the sample was 20 µl at a polymer concentration of 0.2% (w / v). The molecular weights of the polymers were determined relative to polystyrene standards. The intrinsic viscosity of PDLLA-PEG-PDLLA in CHDl₃ at 25 ° C it was measured with a Cannon-Fenske viscometer.

Thermal analysis of the copolymers was carried out by scanning calorimetry (DSC) using a TA Instruments 2000 controller and DuPont 910S (Newcastle, Delaware). The heating rate was 10 ° C / minute and the copolymer and paclitaxel / copolymer matrix samples were weighed (3-5 mg) into open and corrugated aluminum sample trays.

H nuclear magnetic resonance was used¹ (ΚΜΝ) to determine the chemical composition of the polymer. The ^^ ΜΝ spectra of PDLLA-PEG-PDLLA containing paclitaxel were obtained in CDCl₃ using an NMR instrument (Bruker, AC-200E) at 200 ΜΙΙ /. The polymer concentration was 1-2%.

The morphology of the paclitaxel / PDLLA-PEG-PDLLA paste was investigated using scanning electron microscopy (8ΒΜ) (Hitachi F-2300). The sample was coated with 60% Au and 40% Pd (10-15 nm thickness) using a Hummer instrument (Technics, USA).

D. In vitro release of paclitaxel

A small granule of PDLLA paste: PCL containing 20% ​​paclitaxel (approximately 2 mg) or a cylinder (prepared by extruding molten paste through a syringe) of PDLLA-PEG-PDLLA paste containing 20% ​​paclitaxel They were placed in stoppered 14 ml glass tubes containing 10 ml of phosphate buffered saline (pH 7.4) with 0.4 g / l albumin. The tube was incubated at 37 ° C with gentle rotary mixing. The supernatant was periodically removed for paclitaxel analysis and replaced with buffer.

ES 2 207 451 T3

Fresh PBS / albumin. The supernatant (10 ml) was extracted with 1 ml of methylene chloride. The aqueous phase was separated by decantation and the methylene chloride phase was dried under a stream of nitrogen at 60 ° C. The dried residue was reconstituted in a 40:60 mixture of water: acetonitrile and centrifuged at 10,000 g for approximately 1 minute. The amount of paclitaxel in the supernatant was then analyzed by HPLC. HPLC analysis was performed using a 110A pump and a C-8 Utrasphere column (Beckman) and a SPD-6A UV detector set at 232 nm, a SIL-9A autoinjector, and a C-R3A integrator (Shimadzu). The injection volume was 20 µl and the flow rate was 1 ml / minute. The mobile phase was 58% acetonitrile, 5% methane, and 37% distilled water.

E. Results and explanation

The molecular weight and molecular weight distribution of PDLLA-PEG-PDLLA, relative to polystyrene standards, was measured by GPC (Figure 30). The intrinsic viscosity of the copolymer in CHCl3 at 25 ° C was determined using a Canon-Fenske viscometer. The molecular weight and intrinsic viscosity decreased with increasing PEG content. The polydispersities of PDLLA-PEG-PDLLA with PEG content of 10% -40% were 2.4 to 3.5. However, the 70% PEG copolymer had a narrow molecular weight distribution with a polydispersity of 1.21. This may be due to a high PEG content that reduces the possibility of side reactions such as transesterification, which result in a broad molecular weight distribution of the polymer. Alternatively, a rolled structure of the hydrophobic-hydrophilic block copolymers can result in an artificially low value of polydispersity.

DSC scans of pure PEG and PDLLA-PEG-PDLLA copolymers are provided in Figures 25 and 26. PEG and PDLLA-PEG-PDLLA with PEG contents of 70% and 40% showed endothermic peaks with enthalpy and temperature decreasing as the PEG content of the copolymer decreased. The endothermic peaks in the 40% and 70% PEG copolymers were probably due to melting of the PEG zone, indicating the occurrence of phase separation. Although pure PEG had a sharp melting point, copolymers of both 70% and 40% PEGs showed broad peaks with different kink in the case of 70% PEG. The broad melting peaks have resulted from PDLLA interference with PEG crystallization. The kinking in the case of 70% PEG may represent the glass transition of the PDLLA zone. No thermal changes occurred in the copolymers with PEG content of 10%, 20% and 30% in a temperature range of 10250 ° C, indicating that no significant crystallization had occurred (therefore, it may be the separation of phases).

DSC thermograms of PDLLA: PCL mixtures (70:30, 80:20, 90:10) without paclitaxel or with 20% paclitaxel showed an endothermic peak at approximately 60 ° C, resulting from the melting of PCL. Due to the amorphous nature of PDLLA and its low molecular weight (800), fusion and glass transitions of PDLLA were not observed. No thermal changes due to recrystallization or melting of paclitaxel were observed.

PDLLA-PEG-PDLLA copolymers of 20% and 30% PEG content were selected as optimal formulation materials for the paste for the following reasons: A PDLLA-PEG-PDLLA of 10% PEG could not be melted at a temperature of about 60 ° C; the 40% and 70% PEG copolymers melted easily at 60 ° C and the 20% and 30% PEG copolymer became a viscous liquid between 50 ° C and 60 ° C; and the swelling of the 40% and 70% PEG copolymers in water was very high, leading to a rapid dispersion of the pastes in water.

The in vitro release profiles of paclitaxel from PDLLA-PEG-PDLLA cylinders are shown in Figure 27. The experiment measuring release from 40% PEG cylinders was terminated as the cylinders had a grade very high swelling (approximately 200% water absorption in one day) and disintegrated in a few days. The fraction of paclitaxel released from the 30% PEG cylinders increased gradually over 70 days. The fraction released from the 20% PEG cylinders increased slowly up to 30 days and then increased abruptly, which was followed by another period of gradual increase. There was a significant difference in the extent to which each individual cylinder (20% PEG9 content showed the abrupt change in paclitaxel release. Before the steep rise, the paclitaxel release fraction was lower for copolymers with lower PEG content for the same cylinder diameter (1 mm). The 40% and 30% PEG cylinders showed much higher paclitaxel release rates than the 20% PEG cylinders. For example, the 30% PEG cylinder released 17% paclitaxe in 30 days, compared to a 2% release from the 20% PEG cylinder. Smaller diameter cylinders resulted in higher release rates (for example, in 30 days, 30% PEG cylinders with 0.65 mm and 1 m diameters released 26% and 17% paclitaxel, respectively (Figure 27)).

The above observations can be explained by the release mechanisms of paclitaxel from the cylinders. Paclitaxel was dispersed in the polymer in the form of crystals, as observed by light microscopy. The crystals began to dissolve in the copolymer matrix at 170 ° C and completely dissolved at 180 ° C, as DSC thermograms by hot-stage microscopy of PDLLA-PEG-PDLLA paste containing 20% ​​paclitaxel (30 % PEG) showed a small recrystallization exotherm (16 J / g, 190 ° C) and a melting exotherm (6 J7g, 212 ° C) for paclitaxel (Figure 25), indicating recrystallization of paclitaxel from the copolymer melt after 180 ° C. In this type of drug / polymer matrix, paclitaxel could be released through diffusion and / or polymer erosion.

ES 2 207 451 T3

In the case of controlled diffusion, the drug can be released by molecular diffusion into the polymer and / or through the open channels formed by the connected drug particles. Therefore, for a content of 20%, some paclitaxel particles were isolated and the paclitaxel can be released by dissolution in the copolymer followed by diffusion. Other paclitaxel particles could form aggregates that connect to the surface and be released through diffusion through the channels. In both cases, cylinders with a smaller dimension provided faster drug release due to the shorter diffusion path (Figure 27).

The changes in dimensions and the water absorption of the cylinders were recorded during the release (Figure 28). The changes in length, diameter, and wet weight of the 30% PEG cylinders increased rapidly to a maximum in 2 days, remained unchanged for approximately 15 days, and then gradually decreased. The initial diameter of the cylinder was not affected by the swelling behavior. For the 20% PEG cylinder, the length decreased by 10% in one day and leveled off, while the diameter and water absorption gradually increased over time. As more PEG in the copolymer absorbed more water to facilitate diffusion of paclitaxel, faster release was observed (Figure 27).

The molecular weight degradation of the PDLLA-PEG-PDLLA pulp copolymer was verified by GPC. For the 20% PEG cylinder, the editing volume at the peak position increased with time, indicating a reduced polymer molecular weight during the course of the release experiment (Figure 30). A biphasic molecular weight distribution was observed on day 69. The polymer molecular weight also decreased for the 30% PEG cylinders (1mm and 0.65mm). However, no biphasic distribution was observed.

NMR spectra revealed a PEG peak at 3.6 ppm and PDLLA peaks at 1.65 ppm and 5.1 ppm. The peak area of ​​PEG relative to PDLLA in the copolymer decreased significantly after 69 days (Figure 29), indicating dissolution of PEG after its dissociation from PDLLA. The loss of dry mass of the cylinders was also recorded (Figure 29) and shows a decreasing degradation rate of the order of 30% of PEG - 0.65 mm> 30% of PET - 1 mm> 20% of PEG - 1 mm .

Morphological changes of the dry casts before and during the release of paclitaxel were observed using SEM (Figure 31). Briefly, solid paclitaxel crystals and non-porous polymeric matrices were observed prior to release (Figures 31A and 31B). After 69 days of release, no paclitaxel crystals were observed and the matrices contained many pores due to polymer degradation and water absorption (Figures 31C and 31D).

The 30% PEG cylinders showed extensive swelling after only two days in water (Figure 28) and thus, the diffusion obstruction of the detached water-soluble PEG block and the degraded PDLLA (i.e. , DL-lactic acid oligomers). As the loss of mass and degradation of the 30% PEG cylinders were continuous, the contribution of the erosion release increased gradually resulting in a sustained release of paclitaxel without any abrupt change (Figure 27). For the 20% PEG cylinders, swelling was low initially (Figure 28) resulting in slow diffusion of degradation products. Therefore, the degradation products in the inner zone were first retained, while there were less degradation products in the outer zone due to the short diffusion path, the degradation products accelerated the degradation rate, since the end groups of carboxylic acid in the oligomers catalyzed hydrolytic degradation. This resulted in a high molecular weight shell and a low molecular weight interior, as indicated by the biphasic molecular weight distribution of the copolymers (Figure 30, day 69). As shell breakage depended on factors such as shell strength, thickness and defects and interior degradation products, the onset and extent of loss of interior degradation products were highly variable. As the shell breakage was not congruent and the drug in the polymer was not microscopically homogeneous, the value of time for a burst of release and the extent of a burst were different for the four samples tested (Figure 27).

The release of paclitaxel from mixtures of PDLLA and PCL and pure PCL are shown in Figure 32. Briefly, the fraction released increased with the content of PDLLA in the mixture. For example, in 10 days, the paclitaxel released from PDLLA: PCL 80:20, 70:30, and 0: 100 was 17%, 11%, and 6%, respectively. After an initial burst in one day, an approximately constant release of PDLLA: PCL 80:20 pulp was obtained. No significant degree of swelling was observed during release. For PDLLA: PCL mixtures, since PDLLA had a very low molecular weight of about 800, it was rapidly hydrolyzed to water soluble products without a long delay in mass loss. PCL was noted as the "support" material to prevent the paste from rapidly disintegrating. Therefore, the release rate increased with the content of PDLLA in the mixture due to increased degradation. Continuous erosion of PDLLA controlled the release of paclitaxel and resulted in a steady release. The release of paclitaxel from pure PCL was probably diffusion controlled due to the low degradation rate (within 1-2 years) of PCL.

Difficulties were found in the PDLLA: PCL 90:10 pulp release study with 20% paclitaxel content due to disintegration of the pulp granulate in 24 hours of incubation. Briefly, during the first 12 hours of incubation, the samples were taken every hour in order to ensure the falling conditions for the release of paclitaxel. The paclitaxel released from the 90:10 paste was 25-35% in 10 hours.

PDLLA 90:10 paste: PCL containing 30% paclitaxel released more paclitaxel than PD37 paste

ES 2 207 451 T3

ALL: PCL 90:10 containing 20% ​​paclitaxel. Thus, modulation of the release rate of paclitaxel, which was regulated by the properties of the polymer and chemotherapeutic agents, as well as the site of administration, was important in the development of local therapy.

Example 21

Preparation of polymer compositions containing water-soluble additives and paclitaxel

A. Preparation of polymeric compositions

Microparticles of paclitaxel / additive co-precipitates were prepared and subsequently added to PCL for paste forms. Briefly, paclitaxel (100 mg) was dissolved in 0.5 ml of ethanol (95%) and mixed with the additive (100 mg) previously dissolved or dispersed in 1.0 ml of distilled water. The mixture was ground until a smooth paste was formed. The paste was spread in a Petri dish and air dried overnight at 37 ° C. The dry mass was pulverized using a hammer mortar and passed through a 140 mesh (106 µm) sieve (Endecotts Test Sieves Ltd, London, England). The microparticles (40%) were then incorporated into molten PCL (60%) at 65 ° C corresponding to a content of 20% of paclitaxel. The additives used in the study were gelatin (type B, category 100, Fisher Scientific, methylcellulose (British Drug Houses), dextran, T500 (Pharmatia, Sweden), albumin (Fisher Scientific) and sodium chloride (Fisher Scientific). Paclitaxel and gelatin or albumin microparticles were prepared as described above, but passed through a 60 mesh (270 μm) sieve (Endecotts Test Sieves Ltd, London, England) to assess the effect of the size of the microparticles on the release of paclitaxel from the paste. The pastes were also prepared to contain 10, 20, or 30% gelatin and 20% paclitaxel in PCL to study the effect of the additive ratio on drug release. Unless otherwise specified, pastes containing 20% ​​paclitaxel dispersed in PCL were prepared to serve as controls for release rate studies.

B. Drug release studies

A granulate of approximately 2.5 mg of paclitaxel-containing paste was suspended in 50 ml of 10 mM PBS (pH 7.4) in screw cap tubes. The tubes were rocked end to end at 37 ° C and at given time intervals 49 ml of supernatant was removed, filtered through a 0.45 µm membrane filter and retained for paclitaxel analysis. An equal volume of PBS was replaced in each tube to maintain immersion conditions throughout the study. For analysis, the filtrates were extracted with 3 x 1 ml of dichloromethane (DCM), the DCM extracts were evaporated to dryness under a stream of nitrogen and redissolved in 1 ml of acetonitrile. Analysis was by HPÑC using a mobile phase of water: methanol: acetonitrile (37: 5: 58) at a flow rate of 1 ml / minute (Beckman Isocratic pump), a C18 reverse phase column (Beckman) and UV detection (Shimadzu SPD A) at 232 mm.

C. Studies of swelling

Paclitaxel / additive / PCL pastes, prepared using microparticles with paclitaxel additive of mesh size # 140 (and # 60 for solar gelatin) were extruded to form cylinders, the pieces were cut, weighed and the diameter and length were measured. of each piece using a micrometer (Mitutoyo Digimatic). The pieces were suspended in distilled water (10 ml) at 37 ° C and at predetermined intervals the water was discarded and the diameter and length of the cylindrical pieces were measured and the samples were weighed. The morphology of the samples (before and after suspension in water) was examined using scanning electron microscopy (SEM) (Hitachi F-2300). The samples were coated with 60% Au and 40% Pd (10-15 nm thickness) using a Hummer Instrument (Technics, USA).

C. Studies of the chick embryo chorioallantoic membrane (CAM)

Fertilized domestic chicken embryos were incubated for 4 days prior to shellless culture. The egg contents were incubated at a relative humidity of 90% and 3% CO2 and on day 6 of incubation, 1 mg of pieces of pasta containing paclitaxel (containing 6 % paclitaxel, 24% gelatin and 70% PCL) or controls (30% gelatin in PCL). After 2 days of exposure, the vascularization was examined using a stereo-microscope interfaced with a video camera; the video signals were then displayed on a computer and printed on video.

E. Results and explanation

The co-precipitated paclitaxel and gelatin or albumin microparticles were hard and brittle and were easily incorporated into PCL, while the other additives produced soft particles that showed a tendency to decomposition during pulping.

Figure 33 shows the time courses of paclitaxel release from pastes containing 20% ​​paclitaxel in PCL or 20% paclitaxel, 20% additive and 60% PCL. The release of paclitaxel from PCL with or without additives followed a biphasic release pattern; initially, there was a higher drug release rate followed by a slower drug release for the drug. The initial period of release rate greater than

ES 2 207 451 T3 paclitaxel from the pulps was believed to be due to dissolution of paclitaxel placed on the surface or diffusion of paclitaxel from the surface areas of the pulp. The later slower phase of the release profiles can be attributed to a decrease in the effective surface area of ​​the drug particles in contact with the buffer, a slow ingress of buffer into the polymer matrix, or an increase in the mean diffusion paths. of the drug through the polymer matrix.

The two phases of the release profiles of paclitaxel from PCL increased in the presence of the hydrophilic additives, of which gelatin, albumin and methyl cellulose produced a greater increase in drug release rates (Figure 33). There was a further increase in paclitaxel release from the polymer matrix when larger paclitaxel-additive particles (270 μm) were used to prepare the paste compared to when smaller paclitaxel-additive particles (106 μm) were used (Figure 34 ). Increases in the amount of additive (eg, gelatin) produced a corresponding increase in drug release (Figure 34). Figure 35A shows the swelling behavior of pastes containing 20% ​​paclitaxel, 20% additive and 60% PCL. The swelling rate followed the order of gelatin> albumin> methylcellulose> dextran> sodium chloride. Furthermore, the swelling rate increased when a higher proportion of the water soluble polymer was added to the paste (Figure 35B). The gelatin or albumin containing pastes swelled rapidly in the first 810 hours, and subsequently the swelling rate decreased when the sample volume change was greater than 40%. The paste prepared using the larger paclitaxel-gelatin particles (270 μ ^ swelled at a higher rate than those prepared with the smaller paclitaxel-gelatin particles (106 μm). All the pastes disintegrated when the volume increased to more than fifty%. SEM studies showed that swelling of the pastes was accompanied by cracking of the matrix (Figure 36). For higher magnifications (Figures 36C and 36D) there was evidence of paclitaxel crystals in the form of needles or rods on the surface of the paste and in close association with gelatin after swelling (Figures 36C and 36D).

Osmotic or swellable hydrophilic agents absorbed as discrete particles into the hydrophobic polymer resulted in drug release by a combination of matrix erosion, drug diffusion through the polymer matrix, and / or diffusion and / or convective flow through the pores created in the matrix by the dissolution of water-soluble additives. Osmotic agents and swellable polymers dispersed in a hydrophobic polymer would be absorbed in water (acting as wetting agents), dissolve or swell and exert turgid pressure that could break the gaps (the polymer layer) between adjacent particles, creating microchannels. and thus facilitating the escape of drug molecules into the surrounding media by diffusion or convective flow. Swelling and cracking of the paste matrix (Figure 36) probably resulted in the formation of microchannels throughout the interior of the matrix. Different rates and extent of polymer swelling (Figure 35) may contribute to differences in observed paclitaxel release rates (Figure 33 and 34).

Figure 37 shows CAMs treated with gelatin-PCL paste (Figure 37A) and 20% paclitaxel-gelatin-PCL paste (Figure 37B). The paste on the surface of the CAMs is shown by the arrows in the Figures. The CAM with the control paste shows a lattice architecture of normal capillary action. CAMs treated with paclitaxel-PCL paste consistently showed vascular regression and areas lacking a capillary network. The incorporation of additives in the paste considerably increased the diameter of the avascular zone (Figure 37).

This study showed that the in vitro release of paclitaxel from PCL could be increased by incorporating paclitaxel / hydrophilic polymer microparticles into the PCL matrix. In vivo studies evaluating the efficacy of the formation to treat subcutaneous tumors in mice also showed that paclitaxel / gelatin / PCL paste significantly reduced tumor mass. Factors such as the type of the water soluble agent, the size of the microparticles and the proportion of the additives were shown to influence the release characteristics of the drug.

Example 22

Procedure to produce nanopastes

Nanopaste is a suspension of microspheres in a hydrophilic gel. In one aspect of the invention, the gel or paste can be spread on tissue as a method of placing drug-containing microspheres close to the target tissue. Being water-based, the paste is rapidly diluted with body fluids causing a decrease in the thickness of the paste and a tendency for the microspheres to be deposited in nearby tissue. An accumulation of drug encapsulated in microspheres is thus placed close to the target tissue.

Reagents and setup used in these experiments include glass beakers, Carbopol 925 (pharmaceutical grade, Goodyear Chemical Co.,), distilled water, sodium hydroxide (1 M) in aqueous solution, sodium hydroxide solution (5 M) in aqueous solution, microspheres in a size range of 0.1 mm to 3 mm suspended in water at 20% w / v (see above).

1. Preparation of cabopol gel at 5% w / v

Sufficient carbopol was added to 1M sodium hydroxide to make a 5% w / v solution. To dissolve the carbopol in the 1M sodium hydroxide, the mixture was allowed to settle for approximately one hour.

ES 2 207 451 T3

During this period of time, the mixture was stirred and, after one hour, the pH was adjusted to 7.4 using 5M sodium hydroxide until the carbopol was completely dissolved. Once a pH of 7.4 was achieved, the gel was converted and allowed to settle for 2 to 3 hours.

two. Procedure to produce nanopaste

A sufficient quantity of microspheres from 0.1 µm to 3 µm was added to water to produce a 20% suspension of the microspheres. Carbopol gel (8 ml of 5% w / v) was placed in a glass beaker and 2 ml of the 20% microsphere suspension was added. The mixture was shaken to completely disperse the microspheres throughout the gel. The mixture was stored at 4 ° C.

Example 23

Paclitaxel complex with cyclodextrins

A. Materials

Paclitaxel was obtained from Hauser Chemical Inc. (Boulder, Colorado). Disodium phosphate (Fisher) citric acid (British Drug Houses), hydroxypropyl-e-cyclodextrin (HP ^ CD), γ-cyclodextrin (γ-CD) and hydroxypropyl-Y-cyclodextrin (HPyCD) were obtained from the company American Maize- Products Company (Hammond, Indiana) and used as received.

B. Methods

1. Solubility studies

Excess amounts of paclitaxel (5 mg) were added to aqueous solutions containing various concentrations of γ-CD, HPy-CD or HP ^ -CD and were gently stirred for approximately 24 hours at 37 ° C. After reaching equilibrium, aliquots of the suspension were filtered through a 0.45 µm membrane filter (Millipore), suitably diluted and analyzed using MPLC. The mobile phase consisted of a mixture of acetonitrile, methanol and water (58: 5:37) at a flow rate of 1.0 ml / minute. The solubility of paclitaxel in a solvent composed of 50:50 water and ethanol (95%) containing various concentrations, up to 10%, of HPe-CD was also investigated. In addition, the dissolution rate profiles of paclitaxel were investigated by adding 2 mg of paclitaxel (as received) to 0, 5, 10, or 20% solutions of HPyCD or 2 mg of previously hydrated paclitaxel (suspending in water for 7 days) to pure water and shaking gently at 37 ° C. Aliquots were taken at various time intervals and assayed for paclitaxel.

two. Stability studies

Solutions containing 20% ​​HPeCD or HPyCD had pH values ​​of 3.9 and 5.2, respectively. The stability of paclitaxel in cyclodextrin solutions was investigated by testing paclitaxel in solutions (20 μg / ml) containing 10 or 20% HPyCD or HPeCD in water or a 50:50 water-ethanol mixture at 37 ° C or 55 ° C, at various time intervals. In addition, the stability of paclitaxel was determined in solutions (1 µg / ml) containing 1%, 2% or 5% HP and ΔCD at 55 ° C.

C. Results

1. Solubility studies

The solubility of paclitaxel increased over the entire range of DC concentration studied; HPeCD produced the largest increase in solubility of paclitaxel (Figure 38). The shape of the solubility curves suggested that the stoichiometries were of a higher order than a 1: 1 complex. Paclitaxel formed Ap-type curves with both HPe-CD and HPy-CD and type A curves_N with γ-CD. The solubility of paclitaxel in a 50% solution of HPe-CD in water was 3.2 mg / ml at 37 ° C, which was an approximately 2,000-fold increase over the solubility of paclitaxel in water. The estimated stability constants (from Figure 39) for first-order paclitaxel-cyclodextrin complexes were 3.1, 5.8, and 7.2 m^-1 for γ-CD, HPy-CD and HPe-CD and the second order complexes were 0.784 x 10³, 1,886 x 10³ and 7,965 x 10³ M^-1 for γ-CD, HPy-Cd and HPe-CD, respectively. The values ​​of the observed stability constants suggested that the inclusion complexes formed by paclitaxel with cyclodextrins were predominantly second order complexes.

The solubility of paclitaxel in a 50:50 water: ethanol mixture increased with an increase in the concentration of cyclodextrin (Figure 40), as observed for complexation in pure water. The apparent stability constant for the complexation of paclitaxel and HPe-CD in the presence of 50% ethanol (26.57 M^-1) was significantly lower (approximately 300 times) than the stability constant in the absence of ethanol. The lower stability constant can be attributed to a change in the dielectric constant or the polarity of the solvent in the presence of ethanol.

Dissolution profiles of paclitaxel in 0, 5, 10 and 20% γ-CD solutions (Figure 41) illustrate the formation of a metastable solution of paclitaxel in pure water or cyclodextrin solutions; the amount of paclitaxel in

ES 2 207 451 T3 solution increased gradually, reached a maximum and subsequently decreased. Dissolution studies using paclitaxel samples that were previously hydrated by suspending them in water for 48 hours did not show the formation of the metastable solution. Furthermore, a DSC analysis of hydrated paclitaxel (vacuum oven dried at room temperature) showed two broad endothermic peaks between 60 and 110 ° C. These peaks were accompanied by a weight loss of approximately 4.5% (determined by thermogravimetric analysis), indicating the presence of hydrate (s). A weight loss of approximately 2.1% would suggest the formation of a paclitaxel monohydrate. Therefore, the appearance of DSC peaks between 60 ° C and 110 ° C and the weight loss of approximately 4.5% suggest the presence of a dihydrate. There was no evidence of endothermic peak (s) between 60 ° C and 110 ° C (DSC results) or weight loss (TGA results) for the paclitaxel samples as received. Therefore the paclitaxel (as received) was anhydrous and in suspension in water it dissolved to form a supersaturated solution which recrystallized as a hydrate of lower solubility (Figure 41).

two. Stability studies

Paclitaxel degradation is dependent on cyclodextrin concentration and followed pseudo-first order degradation kinetics (eg, Figure 42). The degradation rate of paclitaxel in solutions (1 μg / ml of paclitaxel) containing 1% IIP / l-Cl) at 55 ° C are faster (k = 3.38 x 10^-3 h^-1) than the rate at higher concentrations of cyclodextrin. The degradation rate constants of 1.78 x 10^-3 h^-1 and 0.96 x 10^-3h^-1 they were observed for paclitaxel in 10% HP / l-CD and HPy-CD, respectively. Paclitaxel solutions (1 μg / ml) containing 2, 4, 6 or 8% of IIP / l-Cl) did not show any significant difference in the degradation rate compared to those obtained with solutions of 10 or 20% of HP / 1CD (20 μg / ml). The presence of ethanol did not adversely affect the stability of paclitaxel in cyclodextrin solutions.

D. Conclusion

This study showed that the solubility of paclitaxel could be increased by complexation with cyclodextrins. These water-based cyclodextrin formulations can be used in the treatment of various inflammatory diseases.

Example 24

Polymeric compositions with increased concentrations of paclitaxel

PDLLA-MePEG and PDLLA-PEG-PDLLA are block copolymers with hydrophobic (PDLLA) and hydrophilic (PEG and MePEG) regions. At appropriate molecular weights and chemical composition, they can form minute aggregates of the hydrophobic core of PDLLA and the hydrophilic shell of MePEG. Paclitaxel can be introduced into the hydrophobic core, thus providing paclitaxel with increased "solubility".

A. Materials

D, L-lactide was purchased from Aldrich, and stannous octoate, polyethylene glycol (molecular weight 8,000), and MePEG (molecular weight 2,000 and 5,000) were from Sigma. The MePEG (molecular weight 750) was from Union Carbide. The copolymers were synthesized by a ring-opening polymerization process using stannous octoate as a catalyst (Deng et al. J. Polym. Sci., Polym, Lett. 28: 411-416, 1990; Cohn et al, J. Biomed, Mater. Res. 22: 993-1009, 1988).

To synthesize PDLLA-MePEG, a mixture of DL-lactide / MePEG / stannous octoate was added to a 10 milliliter glass ampoule. The ampoule was connected to a vacuum and sealed with a flame. Polymerization was carried out by incubating the ampoule in an oil bath at 150 ° C for 3 hours. To synthesize PDLLA-PEG-PDLLA, a mixture of D, L-lactide / PEG / stannous octoate was transferred to a glass flask, sealed with a rubber stopper and heated for 3 hours in an oven at 150 ° C. The starting compositions of the copolymers are given in Tables 1 and 2. In all cases, the amount of stannous octoate was 0.5% -0.7%.

B. Methods

The polymers were dissolved in acetonitrile and centrifuged at 10,000g for 5 minutes to discard any insoluble impurities. The acetonitrile solution of paclitaxel was then added to each polymer solution to provide a 10 wt% paclitaxel (paclitaxel + polymer) solution. The acetonitrile of the solvent was then removed to obtain a clear matrix of paclitaxel / PDLLA-MePEG, under a stream of nitrogen and heating to 60 ° C. Distilled water, 0.9% NaCl saline or 5% dextrose were added in 4 times the weight of the matrix. The matrix was finally "dissolved" by means of centrifugal mixing and periodic heating to 60 ° C. Clear solutions were obtained in all cases. The particle sizes were all below 50 nm as determined by a submicron particle sizing device (NICOMP model 270). The formulations are provided in Table 1.

ES 2 207 451 T3

TABLE 1

Paclitaxel / PDLLA-MePEG formulations *

<td>PDLLA ^ ePEG</td><td>Solvent media</td><td>Paclitaxel content (final paclitaxel concentrate)</td>

<td> 2000/50/50</td><td>Water</td><td>10% (20 mg / ml)</td>

<td> 2000/40/60</td><td>Water</td><td>10% (20 mg / ml)</td>

<td> 2000/50/50</td><td>Sun. 0.9% saline</td><td>5% (10 mg / ml)</td>

<td> 2000/50/50</td><td>Sun. 0.9% saline</td><td>10% (20 mg / ml)</td>

<td> 2000/50/50</td><td>5% dextrose</td><td>10% (10mg / ml)</td>

<td> 2000/50/50</td><td>5% dextrose</td><td>10% (20 mg / ml)</td>

In the case of PDLLA-PEG-PDLLA (Table 2), since the copolymers cannot dissolve in water, the paclitaxel and the polymer were jointly dissolved in acetone. Water or a water / acetone mixture was gradually added to this paclitaxel-polymer solution to induce the formation of paclitaxel / polymer spheres.

TABLE 2

Composition of PDLLA-PEG-PDLLA

<td>Copolymer name</td><td>PEG Weight (g)</td><td>DL-lactide Weight (g)</td>

<td>PDLLA-PEG-PDLLA 90/10</td><td> 1</td><td> 9</td>

<td>PDLLA-PEG-PDLLA 80/20</td><td> 2</td><td> 8</td>

<td>PDLLA-PEG-PDLLA 70/30</td><td> 3</td><td> 7</td>

<td>PDLLA-PEG-PDLLA 60/40</td><td> 4</td><td> 6</td>

<td>PDLLA-PEG-PDLLA 30/70</td><td> 14</td><td> 6</td>

* PEG molecular weight 8,000.

C. Results

Many of the PDLLA ^ ePEG compositions form clear solutions in water, 0.9% saline, or 5% dextrose, indicating the formation of minute aggregates in the nanometer range. Paclitaxel was successfully introduced into PDLLA ^ ePEG micelles. For example, at% content (this represents 10 mg of paclitaxel in 1 ml of paclitaxel / PDLLA-MePEG / aqueous system) a clear solution of 2000-50 / 50 and 2000-40 / 60 was obtained. The particle size was approximately 60mm.

Example 25

Procedure to produce a movie

The term "films" refers to a polymer formed in one of many geometric shapes. The film can be a thin elastic polymer sheet or a thick 2mm polymer disk, either of which can be applied to the surface of the tissue to prevent subsequent scarring and adhesion formation. This film was designed to be placed on exposed tissue so that any encapsulated drug can be released from the polymer over a long period of time at the tissue site. Films can be prepared by a variety of procedures including, for example, spreading and spraying.

In the spreading technique, the polymer was either melted into a form or dissolved in dichloromethane and poured into a form. The polymer then solidified as it was cooled or solidified as the solvent evaporated, respectively. In the spray technique, the polymer was dissolved in a solvent and sprayed on glass, as the solvent evaporated the polymer solidified on the glass. One pul42

ES 2 207 451 T3 repeated verification made possible the constitution of polymer in the form of a film that can be peeled off the glass.

The reagents and setup that were used in these experiments include a small beaker, a Corning hot plate stirrer, shaping molds (for example, 50 ml centrifuge tube caps), and mold clamping apparatus, vial. 20 ml glass scintillation tube with stopper (plastic insert type), TLC atomizer, nitrogen gas reservoir, polycaprolactone ("PCL" - 10,000 or 20,000 molecular weight; Polysciences), paclitaxel (Sigma, 95% purity), ethanol, ethylene-vinyl acetate ("EVA") (see above), poly (DL) -lactic acid ("PLA" - molecular weight 15,000 to 25,000; Polysciences) and DCM (HPLC grade; Fisher Scientific).

1. Process for Producing Cast-Expansion Films

A small glass beaker with a known weight of PCL was placed in a larger flask containing water (to act as a water bath) and placed on a 70 ° C hot plate until the polymer was completely melted. A known weight of drug was added to the molten polymer and the mixture stirred thoroughly. The molten polymer was poured into a mold and allowed to cool.

two. Procedure to produce films-extension in solvent

A known weight of PCL was placed directly into a 20 ml glass scintillation vial and enough DCM was added to achieve a 10% w / v solution. The solution was mixed and then enough paclitaxel was added to achieve the desired final concentration of paclitaxel. The solution was stirred to dissolve the paclitaxel, allowed to settle for one hour (to decrease the presence of air bubbles), and then slowly poured into a mold. The mold was placed in the fume hood overnight, allowing the DCM to evaporate.

3. Procedure for producing powdered films

A sufficient amount of polymer was weighed directly into a 20 ml glass scintillation vial and enough DCM was added to achieve a 2% w / v solution. The solution was mixed to dissolve the polymer. Using an automatic pipettor, a suitable volume (minimum 5 ml) of the 2% polymer solution was transferred to a separate 20 ml glass scintillation vial. Sufficient paclitaxel was added to the solution and dissolved by shaking the capped vial. To prepare for spraying, the vial cap was removed and the TCL atomizer barrel was immersed in the polymer solution.

The nitrogen reservoir was connected to the gas inlet of the atomizer and the pressure was gradually increased until atomization and spraying began. The molds were sprayed using 5 second oscillating sprays with a 15 second dry time between sprays. Spraying was continued until a suitable thickness of polymer was deposited on the state.

Example 26

Therapeutic-containing polymeric films composed of ethylene-vinyl acetate and a surfactant

Two types of films were investigated in this Example: paclitaxel-containing neat EVA films and paclitaxel-containing EVA / surfactant blends films.

The surfactants that were examined were two hydrophobic surfactants (Span 80 and Pluronic L101) and one hydrophilic surfactant (Pluronic F127). Pluronic surfactants were themselves polymers that had an attractive property in that they could be mixed with EVA to optimize various drug delivery properties. Span 80 is a smaller molecule that is dispersed in the polymer matrix and does not form a mixture.

Surfactants were useful in modulating the release rates of paclitaxel from films and optimizing certain physical parameters of the films. One aspect of the surfactant blend films that indicated that drug release rates could be controlled was the ability to vary the rate and extent to which the compound swelled in water. Diffusion of water in a polymer-drug matrix was critical for drug release from the vehicle. Figures 43C and 43D show the degree of swelling of the films as the level of surfactant in the mixture was altered. The neat EVA films did not swell to any significant extent for 2 months. However, by increasing the level of surfactant added to the EVA, it was possible to increase the degree of swelling of the compound, and by increasing the hydrophobicity, the swelling was increased.

The results of the experiments with these films are shown below in Figures 43A-E. Briefly, Figure 43A shows the release of paclitaxel (in mg) over time from neat EVA films. Figure 43B shows the percentage of drug that remained in the same films. As can be seen from these two figures, as the content of paclitaxel increased (that is, the percentage of paclitaxel by increased weight) the drug release rates increased, showing the dependence of the

ES 2 207 451 T3 expected concentration. As the content of paclitaxel increased, the percentage of paclitaxel remaining in the film also increased, indicating that higher content may be more attractive for long-term release formulations.

The physical strength and elasticity of the films was assessed as shown in Figure 43E. Briefly, Figure 43E shows the stress / strain curves for neat EVA films and EVA / surfactant blends. This raw stress measurement demonstrated that the elasticity of the films was increased with the addition of Pluronic F127, and that the tensile strength (ultimate stress) was increased in a concentration-dependent manner with the addition of Pluronic. F127. Elasticity and strength are important considerations in the design of a film that must be manipulated for particular clinical applications without causing permanent deformation of the composite.

The above data demonstrate the ability of certain density additives to control drug release rates and alter the physical characteristics of the vehicle.

Example 27

Procedure to produce a nano-spray

Nanospray is a suspension of small microspheres in saline solution. If the microspheres are very small (i.e. below 1 μm in diameter) they form a colloid, so the suspension will not settle due to gravity. As described in more detail below, a suspension of 0.1 μm to 1 μm microparticles can be suitably created for aerosol deposition onto tissue directly at the time of surgery (e.g. for vascular adhesions). through laproscopic intervention or through a finger-pumped aerosol (for example, to be delivered topically). Facilities and materials that can be used to produce nano-sprays include a 200 mL water jacketed beaker (Kimax or Pyrex), a Haake circulating water bath, an overhead shaker, and a 2-inch (5.08 cm) in diameter (4-blade stainless steel stirrer-type impeller; Fisher brand), 500 mL glass beaker, hot plate / shaker (Corning brand), 4 x 50 mL polypropylene centrifuge tubes (Nalgene), glass scintillation vials with plastic insert caps, upper centrifuge Benchtop (Beckman), High Speed ​​Centrifuge - Floor Model (JS 21 Beckman), Mettler Analytical Balancer (AJ 100, 0.1 mg), Mettler Digital Top Load Balancer (AE 163, 0.01 mg), Pipettor automatic (Gilson), sterile pipette tips, pumped-action aerosol (Pfeiffer pharmaceuticals) 20 ml, laminar flow hood, PCL (molecular weight 10,000 to 20,000; Polysciences; Warrington, Pennsylvania, USA), EVA "wash" (see below). above), PLA (molecular weight 15,000 to 25,000; Polysciences), polyvinyl alcohol ("PVA" - molecular weight 124,000 to 186,000; 99% hydrolyzed; Aldrich Chemical Co., Milwaukee, WI USA), DCM or "methylene chloride" (HPLC grade, Fisher Scientific), distilled water and sterile exiting solution (Becton and Dickenson or equivalent).

1. Preparation of polymer solutions at 5% (W / V)

Depending on the polymer solution being prepared, the following were weighed directly into a 20 ml glass scintillation vial: 1.00 g of PCL or PLA or 0.50 g each of PLA and washed EVA. Using a measuring cylinder, 20 ml of DCM was added and the vial was tightly capped. The vial was allowed to sit at room temperature (25 ° C) until all of the polymer had dissolved.

two. Preparation of stock solution at 3.5% (W / V) of PVA

The solution was prepared by following the procedure provided above, or by diluting the PVA stock solution to 5% (W / V) prepared for the production of microspheres (see Example 28). Briefly, 17.5 g of PVA was weighed directly into a 600 ml glass beaker and 500 ml of distilled water was added. The beaker was capped and placed in a 2,000 ml beaker containing 300 ml of water. The PVA was stirred at 300 rpm at 85 ° C until completely dissolved.

3. Procedure to produce nano-spray

Briefly, 100 ml of the 3.5% PVA solution was placed in the 200 ml water jacketed beaker with a Haake water bath connected. The contents of the beaker were shaken at 3,000 rpm and 10 ml of polymer solution (polymer solution used based on the type of nano-spray being produced) was pipetted into the stirred PVA over a period of 2 minutes, using a 5 ml automatic pipettor. After 3 minutes, the stirring speed was adjusted to 2,500 rpm (+/- 200 rpm) for 2.5 hours. After 2.5 hours the stirring paddle was removed from the nano-spray preparation and rinsed with 10 ml of distilled water, allowing the rinse solution to flow to the nano-spray preparation.

The microsphere preparation was poured into a 500 ml beaker. The jacketed water bath was washed with 70 ml of distilled water, allowing the 70 ml of rinse solution to go to the microsphere preparation. The 180 ml microsphere preparation was shaken with a glass rod and also poured into four 50 ml polypropylene centrifuge tubes, which were centrifuged at 10,000 g (+/- 1,000 g) for the duration44

ES 2 207 451 T3 you 10 minutes. The PVA solution was removed from each microsphere sedimentation and discarded. Distilled water (5 ml) was added to each centrifuge tube and centrifuged. The four microsphere suspensions were pooled in a centrifuge tube using 20 ml of distilled water and centrifuged for 10 minutes at 10,000 g (+/- 1,000 g). The supernatant was removed from the microsphere pellet and 40 ml of distilled water was added, and the microsphere preparation was centrifuged (this procedure was repeated 3 times). The microsphere preparation was then transferred to a preweighed glass scintillation vial.

The vial was allowed to sit for 1 hour at room temperature (25 ° C) to allow the 2 µm and 3 µm diameter microspheres to settle under the action of gravity. After 1 hour, the upper 9 ml of the suspension was removed, placed in a sterile capped 50 ml centrifuge tube and centrifuged at 10,000 g (+/- 1,000 g) for 10 minutes. The supernatant was discarded and the pellet was resuspended in 20 ml of sterile saline by centrifuging the suspension at 10,000 g (+/- 1,000 g) for 10 minutes. The supernatant was discarded and the pellet was resuspended in sterile saline. The amount of saline used depended on the final concentration required for the suspension (usually 10% w / v). The nanospray suspension was added to the aerosol.

Example 28

Manufacture of microspheres

The facility used for the manufacture of microspheres includes: 200 ml water jacketed beaker (Kimax or Pyrex), Haake circulating water bath, overhead stirrer and 2 inch diameter controller (propeller type stainless steel stirrer, 4 paddles, Fisher brand), 500 mL glass beaker, hot plate / shaker (Corning brand), 4 x 50 mL polypropylene centrifuge tubes (Nalgene), scintillation vials with plastic insert caps, Table Top Centrifuge (GPR Beckman), High Speed ​​Centrifuge, Floor Model (JS 21 Beckman), Mettler Analytical Balancer (AJ 100, 0.1 mg), Mettler Digital Top Load Balancer (AE 163, 0.01 mg ), and automatic pipettor (Gilson). Reagents include PCL (molecular weight 10,000 to 20,000; Polysciences, Warrington, Pennsylvania, USA), EVA "wash" (see "wash" method below), PLA (molecular weight 15,000 to 25,000; POlysciences), poly (vinyl alcohol) ("PVA" - molecular weight 124,000 to 186,000; 99% hydrolyzed; Aldrich Chemical Co. Milwaukee, WI, USA), DCM or "methylene chloride"; HPLC grade, Fisher Scientific and distilled water.

A. Preparation of 5% (w / v) polymer solutions

DCL (1.00 g) or PLA, 0.50 g each of PLA and washed EVA were weighed directly into a 20 ml glass scintillation vial. Then 20 milliliters of DCM were added. The vial was capped and stored at room temperature (25 ° C) for one hour (occasional shaking can be used) or until all of the polymer dissolved. The solution can be stored at room temperature for at least two weeks.

B. Preparation of stock solution at 5% (w / v) of PVA

25 grams of PVA was weighed directly into a 600 ml glass beaker and 500 ml of distilled water was added along with a 3 inch (7.62 cm) Teflon coated stir bar. The beaker was capped with glass to reduce evaporative losses and placed in a 2,000 ml glass beaker containing 300 ml of water. The PVA was stirred at 300 rpm at 85 ° C (Corning hot plate / shaker) for 2 hours or until completely dissolved. Dissolution of the PVA was determined by visual verification; the solution must be clear. The solution was then transferred to a glass screw cap storage container and stored at 4 ° C for up to two months. However, the solution must be warmed to room temperature before use or dilution.

C. Procedure to produce microspheres

Based on the size of the microspheres being prepared (see Table 1), 100 ml of PVA solution (concentrations given in Table 1) was placed in the 200 ml water jacketed beaker. A Haake circulating water bath was connected to this beaker and the contents were allowed to equilibrate at 27 ° C (+/- 1 ° C) for 10 minutes. Based on the size of the microspheres being prepared (see Table 1), the overhead stirrer starting speed was adjusted and the overhead stirrer blade was placed midway below the PVA solution. The shaker was started and then 10 ml of polymer solution (polymer solution used based on the type of microspheres being produced) was dripped into the stirred PVA over a period of 2 minutes using a 5 minute automatic pipettor. ml. After 3 minutes, the stirrer speed was adjusted (see Table 1) and the solution was stirred for an additional 2.5 hours. The stirring paddle was then removed from the microsphere preparation and rinsed with 10 ml of distilled water so that the rinse solution was purged into the microsphere preparation. The microsphere preparation was then poured into a 500 ml beaker and the jacketed water bath was washed with 70 ml of distilled water, which was also allowed to drain into the microsphere preparation. The 180 ml microsphere preparation was then shaken with a glass rod and equal amounts were poured into four 50 ml polypropylene centrifuge tubes. The tubes were then capped and centrifuged for

ES 2 207 451 T3 minutes (force given in Table 1). 24 milliliters of the PVA solution were removed from each microsphere pellet.

TABLE 1

PVA concentrations, stirring speeds, and centrifugal force requirements for each range of microsphere diameters

<td rowspan="2">PRODUCTION STAGE</td><td colspan="3">MICROSPHERES DIAMETER INTERVALS</td>

<td>30 μτη to 100 μιτι</td><td>10 μπι to 3 0 μιη</td><td>0.1 μιη to 3 μτη</td>

<td>Concentration of PVA</td><td>2.5% (w / v) (namely, mother 5% diluted with distilled water)</td><td>5% (p / v) (namely, mother without dilute)</td><td>3.5% (w / v) j (i.e. 1 mother at 5% I diluted with | distilled water)</td>

<td>Speed ​​of starting shaking</td><td>500 rpm +/- 50 rpm</td><td>500 rpm +/- 50 rpm</td><td>3000 rpm +/- 200 rpm</td>

<td>Speed ​​of tight shaking</td><td>500 rpm +/- 50 rpm</td><td>500 rpm +/- 50 rpm</td><td>2500 rpm +/- 200 rpm</td>

<td>Centrifugal force</td><td>1000 g +/- 100 g (Model table higher)</td><td>1000 g +/- 100 g (Model table higher)</td><td>10,000 g +/- 100 g (Model velocity elevated)</td>

5 milliliters of distilled water were then added to each centrifuge tube and centrifuged to resuspend the microspheres. The four microsphere suspensions were then pooled in a centrifuge tube together with 20 ml of distilled water and centrifuged for another 10 minutes (force given in Table 1). This procedure was repeated two additional times for a total of three washes. The microspheres were then centrifuged one final time and resuspended in 10 ml of distilled water. After the final wash, the microsphere preparation was transferred to a preweighed glass scintillation vial. The vial was capped and left overnight at room temperature (25 ° C) in order to allow the microspheres to settle under the action of gravity. Since microspheres falling in the size range 0.1 µm to 3 µm did not settle under the action of gravity, they were left in the 10 ml suspension.

ES 2 207 451 T3

D. Drying 10 μm to 30 μm or 30 μm to 100 μm diameter microspheres

After the microspheres settled at room temperature overnight, the supernatant was removed from the settled microspheres. The microspheres were allowed to dry in the uncapped vial in a drawer for a period of one week or until completely dry (vial at constant weight). Faster drying can be accomplished by leaving the vial uncapped under a slow stream of nitrogen gas (flow of approximately 10 ml / minute) in the fume hood. When completely dry (constant weight vial), the vial was weighed and capped. The capped and labeled vial was stored at room temperature in a drawer. Microspheres were normally stored for no more than 3 months.

E. Determination of the concentration of the suspension of microspheres from 0.1 μm to 3 μm in diameter

This range of microsphere sizes was not separated by sedimentation, so they were left in suspension at 4 ° C for up to four weeks. To determine the concentration of microspheres in the 10 ml suspension, a 200 µl sample of the suspension was pipetted into a 1.5 ml preweighed microcentrifuge tube. The tube was then centrifuged at 10,000 g (Eppendorf bench top microcentrifuge), the supernatant was removed and the tube was allowed to dry at 50 ° C overnight. The tube was then weighed again in order to determine the weight of dry microspheres in the tube.

F. Preparation of microspheres containing paclitaxel

In order to prepare microspheres containing paclitaxel, a quantity of heavy paclitaxel (based on the percentage of paclitaxel to be encapsulated) was placed directly into a 20 ml glass scintillation vial. 10 milliliters of an appropriate polymer solution was then added to the vial containing the paclitaxel, which was then centrifuged until the paclitaxel dissolved.

Microspheres containing paclitaxel can then be produced essentially as described above in steps (C) to (E).

Example 29

Surfactant Coated Microspheres

A. Materials and methods

Microspheres were made from poly (DL-lactic acid) (PLA), poly (methyl methacrylate) (PMMA), polycaprolactone (PLC), and 50% ethylene-vinyl acetate (EVA): PLA essentially as described with anteriority. The size ranged from 10 to 100 µm with a mean diameter of 45 µm.

Human blood was obtained from healthy volunteers. Neutrophils (white blood cells) were separated from the blood using dextran sedimentation and Ficoll Hypaque centrifugation techniques. Neutrophils were suspended at 5 million cells per ml in HBSS.

Neutrophil activation levels were determined by the generation of reactive oxygen species as determined by chemiluminescence. In particular, chemiluminescence was determined using a LKB luminometer with a 1 µΜ luminol enhancer. The plasma pre-coating (or oxonization) of the microspheres was performed by suspending 10 mg of microspheres in 0.5 ml of plasma and shaking at 37 ° C for 30 minutes.

The microspheres were then washed with 1 ml of HBSS and the pellet of centrifuged microspheres was added to the neutrophil suspension at 37 ° C for a time t = 0. The surfaces of the microspheres were modified using a surfactant called Pluronic F127 (BASF) suspending 10 mg of microspheres in 0.5 ml of 2% w / w solution of F127 in HBSS for 30 minutes at 37 ° C. The microspheres were then raised twice in 1 ml of HBSS before adding to neutrophils or plasma for additional pre-coating.

B. Results

Figure 44 shows that the untreated microspheres provide chemiluminescence values ​​of less than 50 mV. These values ​​represent low levels of neutrophil activation. By way of comparison, inflammatory microcrystals can provide values ​​close to 1,000 mV, soluble chemical activators can provide values ​​close to 5,000 mV. However, when the microspheres are previously coated with plasma, all chemiluminescence values ​​are amplified up to the range of 100 to 300 mV (Figure 44). These levels of neutrophil response or activation can be considered mildly inflammatory. PMMA provided the greatest response and could be considered the most inflammatory. PLA and PCL were both three to four times more potent in activating neutrophils after plasma pretreatment (or oxonization), but there is little difference between the two polymers in this regard. EVA: PLA is not likely to be used in angiogenesis formulations as the microspheres are difficult to dry and are resuspended in aqueous buffer. This effect of plasma is called oxonization and results in the adsorption of antibodies or molecules.

Complementary ES 2 207 451 T3 on the surface. These absorbed species interact with receptors or white blood cells and cause amplified cellular activation.

Figures 45-48 describe the effects of plasma pretreatment of PCL, PMMA, PLA, and EVA: PLA, and also show the effect of a Pluronic F127 coating prior to plasma pre-coating of the microspheres. These figures all show the same effect: (1) plasma precoating amplifies the response; (2) precoating with Pluronic F127 has no effect on its own; (3) the amplified neutrophil response caused by precoating with plasma can be strongly inhibited by pretreating the surface of the spheres with 2% Pluronic F127.

The nature of the protein species adsorbed from plasma was also studied by electrophoresis. Using this method, it was shown that pretreatment of the polymeric surface with Pluronic F127 inhibited the adsorption of antibodies to the polymeric surface.

Figures 49-52 similarly show the effect of precoating microspheres with PCL, PMMA, PLA or EVA: PLA (respectively) with IgG (2 mg / ml) or 2% Pluronic F127 and then IgG (2 mg / ml) . As can be seen from these figures, the amplified response caused by the precoating of microspheres with IgG can be inhibited by treatment with Pluronic F127.

This result shows that by pretreating the polymeric surface of all four types of microspheres with Pluronic F127, the "inflammatory" response of neutrophils to microspheres can be inhibited.

Example 30

Encapsulation of therapeutic agent in poly (e-caprolactone) microspheres. Inhibition of Angiogenesis in the Paclitaxel-Containing Microsphere CAM Assay

This example evaluates the in vitro release rate profile of paclitaxel from biodegradable poly ^ -caprolactone) (PCL) microspheres and demonstrates the in vivo anti-angiogenic activity of paclitaxel released from these microspheres when placed on the CAM.

Reagents used in these experiments include: PCL (molecular weight 35,000-45,000; purchased from Polysciences (Warrington, PA)); DCM from Fisher Scientific Co., Canada; polyvinyl alcohol (PVP) (molecular weight 12,000-18,000, 99% hydrolyzed) from Aldrich Chemical Co. (Milwaukee, Wis.) and paclitaxel from Sigma Chemical Co. (St. Louis, MO). Unless otherwise stated, all chemicals and reagents are used as supplied. Distilled water is used in all of them.

A. Preparation of microspheres

Microspheres were prepared essentially as described in Example 28 using the solvent evaporation method. Briefly, microspheres containing 5% w / w paclitaxel were prepared by dissolving 10 mg of paclitaxel and 190 mg of PCL in 2 ml of DCM, adding 100 ml of 1% PVP aqueous solution and stirring at 1,000 rpm at 25 ° C for 2 hours. The microsphere suspension was centrifuged at 1,000 xg for 10 minutes (Beckman GPR), the supernatants were separated and the microspheres were washed three times with water. The washed microspheres were air dried overnight and stored at room temperature. Control microspheres (in the absence of paclitaxel) were prepared as described above. Microspheres containing 1% and 2% paclitaxel were also prepared. The microspheres were sized using an optical microscope with a staged micrometer.

B. Encapsulation efficiency

A known weight of drug-containing microspheres (approximately 5 mg) was dissolved in 8 ml of acetonitrile and 2 ml of distilled water was added to precipitate the polymer. The mixture was centrifuged at 1,000 g for 10 minutes and the amount of encapsulated paclitaxel was calculated from the absorbance of the supernatant measured in a UV spectrophotometer (Hewlett-Packard 8452A diode array spectrophotometer) at 232 nm.

C. Drug release studies

Approximately 10 mg of paclitaxel-containing microspheres were suspended in 20 ml of 10mM PBS (pH7.4) in ring-capped tubes. The tubes were turned from one extreme to another at 37 ° C and at given time intervals 19.5 ml of supernatant was removed (after allowing the microspheres to settle to the bottom), filtered through a 0.45 μm membrane filter and retained. for a paclitaxel test. An equal volume of PBS was returned to each tube to maintain immersion conditions throughout the study. The filtrates were extracted with 3 x 1 ml of DCM, the DCM extracts were evaporated to dryness using a stream of nitrogen, redissolved in 1 ml of acetonitrile and analyzed by HPLC using a mobile phase of water: methanol: acetonitrile (37: 5: 58) at a flow rate of 1 ml / minute (Beckman isocratic pump), a C8 reversed phase column (Beckman) and UV detection (Shimadzu SPD A) at 232 nm.

ES 2 207 451 T3

D. CAM studies

Fertilized domestic chicken embryos were incubated for 4 days before culturing in the absence of a shell. On day 6 of incubation, 1 mg aliquots of microspheres containing 5% paclitaxel or control (paclitaxel-free) were placed directly on the surface of the CAM. After a 2 day exposure, vascularization was examined using a stereomicroscope interfaced with a video camera; the video signals were then displayed on a computer and printed on video.

E. Scanning electron microscopy

Microspheres were placed in sample holders, sputter coated with gold and then placed in a Philips 501B SEM device operating at 15 kV.

F. Results

The size range for the microsphere samples was between 30-100 µm, although there was evidence in all batches of paclitaxel-containing microspheres or controls for some microspheres that fell outside this range. The efficiency of including a content of PCL microspheres with paclitaxel was always greater than 95% for all drug contents studied. Scanning electron microscopy showed that the microspheres were all spherical and many exhibited rough or pitted surface morphology. There did not appear to be any evidence of solid drug on the surface of the microspheres.

The time courses of paclitaxel release from microspheres with PCL content of 1%, 2% and 5% are shown in Figure 53A. The release rate profiles were biphasic. There was a rapid initial release of paclitaxel or "surge phase" for all drug contents. The surge phase occurred on 1-2 days for a paclitaxel content of 1% and 2% and on 3-4 days for microspheres with a 5% content. The initial rapid release phase was followed by a significantly slower drug release phase. For microspheres containing 1% or 2% paclitaxel there was no additional drug release after 21 days. For a paclitaxel content of 5%, the microspheres had released approximately 20% of the total drug content after 21 days.

Figure 53B shows CAMs treated with control PCL microspheres, and Figure 53C shows treatment with microspheres containing 5% paclitaxel. CAM with the control microspheres showed a normal capillary network architecture. CAM treated with paclitaxel-PCL microspheres showed considerable vascular regression and areas that were devoid of a capillary network.

G. Explanation

The solvent evaporation method to make paclitaxel-containing microspheres produced very high paclitaxel encapsulation efficiencies between 95-100%. This was due to the poor solubility in water of paclitaxel and its hydrophobic nature favored the division into parts of the organic solvent phase containing the polymer.

The biphasic release profile for paclitaxel was typical of the release pattern for many drugs from biodegradable polymeric matrices. Poly ^ -caprolactone) is an aliphatic polyester that can be degraded by hydrolysis under physiological conditions and is non-toxic and compatible with fabrics. The degradation of PCL is significantly slower than that of the intensively investigated polymers and copolymers of lactic and glycolic acids and is therefore suitable for the design of long-term drug delivery systems. The initial rapid or surge phase of release of paclitaxel was believed to be due to diffusion release of the drug from the surface area of ​​the microspheres (close to the surface of the microsphere). The release of paclitaxel in the second (slower) phase of the release profiles was probably not due to degradation or erosion of PCl because studies have shown that, under in vitro conditions in water, there was no significant weight loss. or surface erosion of PCL over a period of 7.5 weeks. The slower release phase of paclitaxel was probably due to dissolution of the drug in the fluid-filled pores in the polymer matrix and diffusion through the pores. The higher release rate at a higher content of paclitaxel was probably a consequence of a more extensive network of pores in the polymer matrix.

Paclitaxel microspheres containing 5% have been shown to release sufficient drug to produce extensive inhibition of angiogenesis when placed on the CAM. Inhibition of blood vessel growth resulted in an avascular zone, as shown in Figure 53C.

Example 31

Manufacture of PLGA microspheres

Microspheres were made from lactic acid-glycolic acid copolymers (PLGA).

ES 2 207 451 T3

A. Method

Microspheres in the size ranges of 0.5 to 10 μm, 10-20 μm, and 30-100 μm were made using standard methods (the polymer was dissolved in dichloromethane and emulsified in a polyvinyl alcohol solution with stirring, as previously described in the PCL or PDLLA microsphere manufacturing methods). Various ratios of PDLLA to GA were used as the polymers are different molecular weights (given as intrinsic viscosity (IV)).

B. Result

Microspheres were successfully made from the following starting polymers:

<td>PLLA</td><td>: GA</td><td>IV</td>

<td> 50</td><td> 50</td><td> 0,74</td>

<td> 50</td><td> 50</td><td> 0,78</td>

<td> 50</td><td> 50</td><td> 1,06</td>

<td> 65</td><td> 35</td><td> 0,55</td>

<td> 75</td><td> 25</td><td> 0,55</td>

<td> 85</td><td> 15</td><td> 0,56</td>

Paclitaxel with contents of 10% or 20% was successfully incorporated into all these microspheres. Examples of the size distributions for a starting polymer (85:15, IV = 0.56) are provided in Figures 5457. Paclitaxel release experiments were performed using microspheres of various sizes and various compositions. Release rates are shown in Figures 58-61.

Example 32

Paclitaxel encapsulation in nylon microcapsules

Therapeutic agents can also be encapsulated in a wide variety of carriers that can be formed into a selected form or device. For example, as described in more detail below, paclitaxel can be incorporated into nylon microcapsules that can be formulated in the form of artificial heart valves, vascular grafts, surgical nets, or sutures.

A. Preparation of microcapsules containing paclitaxel

Paclitaxel was encapsulated in nylon microcapsules using interfacial polymerization techniques. Briefly, 100 mg of paclitaxel and 100 mg of Pluronic F-127 were dissolved in 1 ml of DCM and 0.4 ml (approximately 500 mg) of adipoyl chloride (ADC) was added. This solution was homogenized in 2% PVA solution using a Plytron homogenizer (set to 1) for 15 seconds. A solution of 1,6-hexanediamine (*) in 5 ml of distilled water was added dropwise while homogenizing. The mixture was homogenized for an additional 10 seconds after the addition of the ΙΙΜΙ 'solution. The mixture was transferred to a beaker and stirred with a magnetic stirrer for 3 hours. The mixture was centrifuged, collected, and resuspended in 1 ml of distilled water.

B. Encapsulation efficiency / paclitaxel content

Approximately 0.5 ml of the suspension was filtered and the microspheres were dried. Approximately 2.5 mg of the microcapsules were weighed and suspended in 10 ml of acetonitrile for 24 hours. The supernatant was analyzed for paclitaxel and the result was expressed as a percentage of paclitaxel. Preliminary studies have shown that paclitaxel could be encapsulated in nylon microcapsules with a high content (up to 60%) and a high encapsulation efficiency (greater than 80%).

C. Paclitaxel release studies

Approximately 2.5 mg of paclitaxel-nylon microspheres were suspended in 50 ml of water containing 1 * sodium chloride and urea each and analyzed periodically. The release of paclitaxel from the microcapsules was rapid with more than 95% of the drug released after 72 hours (Figure 62).

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Example 33

Bioadhesive microspheres

A. Preparation of bioadhesive microspheres

Microspheres were prepared from 100 g / mol PLLA with a particle diameter range of 10-60 µm. The microspheres were incubated in a sodium hydroxide solution to produce carboxylic acid groups on the surface by hydrolysis of the polyester. The reaction was characterized with respect to sodium hydroxide concentration and incubation time by measuring surface charge. The reaction reached completion after 45 minutes of incubation in 0.1 M sodium hydroxide. Following the basic treatment, the microspheres were coated with dimethylaminopropylcarbodiimide (DE, a cross-linking agent, by suspending the microspheres in an alcoholic solution of DEC and allowing the mixture to dry as a dispersible powder The weight ratio of microspheres to DEC was 9: 1. After the microspheres were dried, they were dispersed with stirring in a 2% w / v solution of polyacrylic acid (PAA) and the DEC was allowed to react with the PAA to produce a water-insoluble network of cross-linked PAA. on the surface of the microspheres. Scanning electron microscopy was used to confirm the presence of PAA on the surface of the microspheres.

Differential scanning calorimetry of the microspheres before and after base treatment revealed that no changes in volumetric thermal properties (Tg, melting point and degree of crystallinity) were observed by SEM.

B. Paclitaxel release rates in vitro

Microspheres containing paclitaxel (contents of 10% and 30% w / w) were made with the same range of particle diameter sizes and in vitro release profiles for 10 days of release in PBS. Release was proportional to drug content, with 400 µg of paclitaxel released from 5 mg of 30% content microspheres in 10 days and 150 µg released from 10% content microspheres in the same period. The encapsulation efficiency was approximately 80%. The paclitaxel-containing microspheres were incubated in 0.1 M sodium hydroxide for 45 minutes and the zeta potential was measured before and after incubation in sodium hydroxide. The surface change of the paclitaxel-containing microspheres was less than that of the microspheres without paclitaxel both before and after base treatment.

C. Preparation and In Vitro Evaluation of PLLA Coated with Poly-lysine or Fibronectin

PLLA microspheres were prepared containing 1% sudan black to 1% (to color the microspheres). These spheres were suspended in a 2% (w / volume) solution of poly-lysine (sigma Chemical Hydrobromell form) or fibronectin (Sigma) for 10 minutes. The microspheres were washed in buffer once and placed on the inner surface of freshly prepared rat bladders. The bladders were left for 10 minutes and then washed three times in buffer. Residual (bonded) microspheres were present on the bladder wall after the procedure, therefore, bioadhesion (Figures 63A and 63B) was shown to have occurred for both fibronectin and poly-l-lysine coated microspheres.

Example 34 (Reference example)

Evaluation of chronic rejection in an animal model

An accelerated form of atherosclerosis develops in most heart transplant recipients and limits long-term graft survival. The Lewis-F344 heterotopic rat heart transplant chronic rejection model is a useful experimental model because it produces staged atherosclerotic lesions in allografts that survive in the medium and long term. The advantages of the Lewis-F344 model are that: (i) the incidence and severity of atherosclerotic lesions in long-term surviving grafts is quite high; and (ii) an inflammatory stage of the development of the lesion is easily recognized since the system does not require immunosuppression.

Adult male Lewis rats serve as donors and F-344 rats as recipients. Twenty heterotropic abdominal cardiac allografts are transplanted by making a long midline abdominal incision in anesthetized recipients to expose the aorta and inferior vena cava. The two vessels are separated from each other and from the surrounding connective tissue, and small compression forceps are placed on the vessels. Longitudinal incisions (2–3 mm) are made in each vessel at the anastomosis site.

The abdomen of the anesthetized donor rat is opened for the injection of 300 units of aqueous heparin into the inferior vena cava. The chest wall was opened to expose the heart. The vena cavae were ligated, followed by a transection of the ascending aorta and the main pulmonary artery, leaving the origins of the 2 to 3 mm long vessels attached to the heart. The vena cavae distant from the ligatures were divided and the ligature was placed around the mass of the left atrium and the pulmonary veins. The vessels on the lung side of the ligatures were divided and the heart was removed.

The donor heart is placed in the recipient's abdominal cavity and the aortas are sutured together at the

ES 2 207 451 T3 incision site in the recipient vessel. Similarly, the pulmonary artery connects to the incision site of the inferior vena cava in a similar manner. The vessel compression forceps are released (proximal vena cava, distal cava, and proximal aorta and aorta) to minimize bleeding from the needle holes.

Following transplantation, paclitaxel (36%) in polycaprolactone (PCL) paste (n = 10) or PCL paste alone (n = 10) is injected through the epicardium over a length of the outer surface of a coronary artery in 10 rats, so that the area of ​​the artery included in the myocardium remains untreated.

All recipients receive a single intramuscular injection of penicillin G (100,000 units) at the time of graft. Allografts are followed by daily palpitation and their function is rated on a scale of 1 to 4, with 4 representing a normal heart beat and 0 the absence of mechanical activity. Five rats from each group are sacrificed at 14 days and the final five at 28 days. Rats are watched for weight loss and other signs of systemic disease. After 14 or 28 days, the animals are anesthetized and the heart is exposed in the manner of the initial experiment. Coated and uncoated coronary arteries are isolated, fixed in 10% buffered formaldehyde, and examined for histology.

The initial experiment can be modified to use a paclitaxel / EVA film or coated surgical shunts in the coronary arteries following transplantation. The EVA film is applied to the extraluminal surface of the coronary artery in a similar manner as above, while the coated surgical forceps are placed intraluminally.

In addition, these investigations can be further extended to include other organ transplants as well as graft transplants (eg, veins or skin).

Example 35

Evaluation of paclitaxel and other microtubule stabilizing agents for the treatment of nasal polyps

Nasal polyps epithelial cell cultures and / or tissue cultures are used to evaluate the efficacy of formulations containing paclitaxel or other agents in treating nasal polyps. This approach is based on the premise that epithelial cells release cytokines and contribute to chronic inflammation detected in nasal polyposis, as well as rhinitis and asthma, and that an extended-release medication will prevent eosinophilia and inhibit cytokine gene expression. .

Paclitaxel formulations including solutions (the use of cyclodextrins) or suspensions containing paclitaxel encapsulated in microadhesive polymers for use as nasal sprays and / or paclitaxel microencapsulated in mucoadhesive polymers were used as insufflations. These formulations are used in the detailed studies below.

A. Effect of paclitaxel in vitro

Tissue management - Samples of normal nasal mucosa (NM) were obtained from patients without clinical manifestations of rhinitis and a negative prick test during nasal reconstruction surgery. Nasal polyps (PN) samples were obtained from positive and negative skin puncture test patients who had undergone nasal polypectomy. Nasal samples were placed in Ham's F12 medium supplemented with 100 IU / ml of penicillin, 100 µg / ml of streptomycin and 2 µg / ml of amphotericin B and were immediately transported to the laboratory.

Epithelial Cell Culture - NM and NP nasal epithelial cells were isolated by protease digestion as follows. Tissue samples are rinsed 2-3 times with Ham's F12 supplemented with 100 IU / ml penicillin, 100 μg / ml streptomycin and 2 μg / ml amphotericin B and then incubated in a 0.1% protease type XIV in Ham's F12 at 4 ° C overnight. After incubation, 10% FBS is added to neutralize protease activity and epithelial cells are removed by gentle shaking. Cell suspensions are filtered through a 60 mesh cell dissociation sieve and centrifuged at 500 g for 10 minutes at room temperature. The cell pellet is then resuspended in Ham's hormonally defined F12 culture medium (Ham HD) containing the following reagents: 100 IU / ml penicillin, 100 μg / ml streptomycin, 2 μg / ml amphotericin B, 150 μg / ml glutamine, 5 μg / ml transferin, 5 μg / ml insulin, 25 ng / ml epidermal growth factor, 15 μg / ml endothelial cell growth complement, 200 pM triiodothrionine, and 100 hydrocortisone nM. Cell suspensions (10⁵ cells / well) are then plated in collagen-coated wells in 2 ml of Ham's HD and cultured in a humidified atmosphere with 5% CO2 at 37 ° C. The culture medium is changed after one day and then on alternate days. The confluence of cells in monolayers is achieved after 6-10 days of culture.

Generation of Human Epithelial Conditioned Media (HECM) - When epithelial cell cultures reached confluence, Ham's HD was changed to RPMI 1640 medium (Irvin, Scotland) supplemented with 100 IU / ml penicillin, 100 μg / ml streptomycin , 2 μg / ml of amphotericin B, 150 μg / ml of glutamine and 25 mM Hepes buffer (RPMI 10%). The HECM that is generated after 48 hours of incubation with RPMI (10%) is collected from the cultures, centrifuged at 400 g for 10 minutes at room temperature (RT), sterilized by filtration through 0.22 μm filters and stored at -70 ° C until use.

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Eosinophil survival and effect of paclitaxel - Eosinophils are isolated from peripheral blood and the effect of both NM and NP HECM on eosinophil survival is determined in two different ways: time course and dose response analysis. In time-course experiments, eosinophils at a concentration of approximately 250,000 / ml are incubated in six tissue cultures with or without 50% HECM (negative control) and the survival rate is assessed on days 2, 4 , 6 and 8. Other experiments are performed with 1 to 50% HECM. In experiments testing the effect of drugs (e.g., paclitaxel) on HECM-induced eosinophil survival, drug (paclitaxel) 0.1 nM at 10 μ 10 is incubated with eosinophils at 37 ° C for 1 hour before the addition of HECM. In each experiment, negative control wells (culture media only) and positive controls (culture media with HECM) were always titrated. To investigate whether the drugs had any toxic effects, the viability of eosinophils incubated with the drug (various concentrations) is compared to eosinophils cultured with 10% RPMI alone over a 24-hour period.

B. Effect of paclitaxel on cytokine gene expression and release from epithelial cells

Epithelial cells obtained from nasal polyps and normal nasal mucosa are cultured to confluence, and media conditioned with human epithelial cells generated with or without paclitaxel (or other agents) and supernatants are measured by ELISA. Cytokine gene expression is investigated by reverse transcription polymerase chain reaction (RT-PCR) as described by Mullol et al., Clinical and Experimental Allergy 25: 607-615, 1995.

The results show whether paclitaxel modulates cytokine gene expression as a means of inhibiting eosinophil survival. The main disadvantage of using primary cell cultures is that it takes 10 days for cells to reach confluence, dissociating cellular functions from the local environment as well as systemic effects, which would have led to disease in the first place. However, this is an excellent in vitro / ex vivo model to study the growth factors that regulate the function and proliferation of structural cells (eg epithelial cells) and thus elucidate some aspects of mucosal inflammation.

C. Immunological release of chemical mediators from human nasal polyps

Mediated by paclitaxel and other agents - Polyps are obtained at the time of resection and washed 5 times with Tyrode's Buffer fragmented with fine scissors in reproductions of approximately 200 mg wet weight. The reproductions were suspended in 3 ml of buffer containing various concentrations of paclitaxel at 37 ° C and were stimulated (5 minutes later) with 0.2 μg / ml of E antigen. After 15 minutes of incubation with the antigen, the diffused materials are removed from the tissues boiled in fresh buffer for 10 minutes to remove residual histamine. Released histamine and SRS-A are assayed using HPLC.

Example 36

Perivascular administration of agents that disrupt microtubule function

Studies have been conducted to evaluate the efficacy of a paclitaxel-campothecin (PCL) containing surgical paste and / or an EVA film as a perivascular treatment for restenosis.

A. Materials and methods

WISTAR rats weighing 250 to 300 g were anesthetized by intramuscular injection of Innovar (0.33 mg / kg). Once sedated, they were then placed under Halothane anesthesia. After general anesthesia was established, the skin over the neck area was shaved, the piece was fixed and cleaned with betadine. A vertical incision was made over the left carotid artery and the external carotid artery was exposed. Two ligatures were placed around the external carotid artery and a transverse arteriotomy was performed. A number 2 French Fogarty balloon catheter was then introduced into the carotid artery and passed into the left common carotid artery and the balloon was inflated with saline solution. The endothelium was denuded by passing the inflated balloon up and down the carotid artery three times. The catheter was then removed and the ligation was tied in the left external carotid artery.

Rats were randomized into groups of 10 to receive no treatment, polymer alone (EVA film or PCL paste) or polymer plus 20% paclitaxel. The polymer mixture (2.5 mg) was placed in a circumferential fashion around the carotid artery. The wound was then closed. Five rats from each group were sacrificed at 14 and the final 5 at 28 days. Meanwhile, the rats were observed for weight loss or other indications of systemic disease. After 14 or 28 days, the animals were anesthetized and the left carotic artery was isolated, fixed with 10% buffered formaldehyde, and histologically examined.

As a preliminary study, two rats were treated with an EVA film containing 10% campothecin for 14 days to assess the efficacy of campothecin in this disease model.

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B. Results

The results of these studies revealed that polymers containing paclitaxel (20%) completely prevented restenosis, while control animals and animals that received polymer only developed between 28% and 55% luminal compromise at 14 and 28 days after balloon injury (Figures 76A and 76B).

There was absolute inhibition of intimal hyperplasia when paclitaxel was in contact with the vessel wall. However, the effect was very local, as evidenced by the patchy effect of paclitaxel when there was an inability to keep the drug adjacent to the vessel wall (Figures 77A and 77B).

Preliminary data has shown that the campothecin-containing EVA film was effective in preventing the restenoic response in this animal model of the disease. Campothecin completely inhibited intimal hyperplasia in the two animals tested.

Example 37 (Reference example)

Effects of paclitaxel in an animal model of surgical adhesions

The use of a paclitaxel-containing EVA film to reduce adhesion formation in the rabbit cervical model is examined.

Female New Zealand white rabbits were anesthetized and a laparotomy was performed through a midline incision. The uterine necks were exposed and a 5 cm long segment of each was fleshed out using a scalpel. This abrasion is sufficient to remove the serosa, resulting in a punctate hemorrhage. Rabbits are randomly assigned to control or paclitaxel-treated groups and post-operative evaluation periods of two, four, and eight weeks. In the paclitaxel-treated group, each cervix is ​​completely covered with a paclitaxel-containing EVA film following abrasion. The musculo-peritoneal layer is closed with sutures and the cutaneous layer with skin staples.

Animals were evaluated for adhesion formation two, four, or eight weeks after surgery. The animals were humanely euthanized and necropsies were performed. The cervixes were examined grossly and histologically using standard microscopic techniques. Roughly, adhesions were graded using a standard scoring system that is based on the fact that 5 cm of the cervix is ​​traumatized; therefore, the extent of adhesion formation is determined by measuring the length of the area containing adhesions. The following grading system is used: 0 = no adhesion, 1 = adhesion in 25% of the area, 2 = adhesion in 50% of the area and 3 = total adhesion included. The severity of adhesions is measured as follows. 0 = no resistance to separation, 0.5 = some resistance (moderate force required), and 1 = sharp dissection required. Total grade is additive, with a range of adhesion scores of 0-4 representing both extent and severity.

Example 38 (Reference example)

Effect of paclitaxel in an animal model of systemic lupus erythematosus

The efficacy of paclitaxel in systemic lupus erythematosus is determined by treating female NZB / NZW F1 (B / W) mice with micellar paclitaxel. This strain of mice develops a disease similar to human SLE. At one month of age, these mice have an elevated level of vessel B cells that spontaneously secrete immunoglobulin, compared to normal mice. Elevated levels of anti-ssDNA antibody occur by two months of age. By 5 months of age, immunoglobulin accumulates along the gromerular capillary walls. Severe glomerulonephritis evolves and by 9 months of age, 50% of B / W mice are dead.

A. Materials and methods

Female B / W mice are purchased from The Jackson Laboratory (Bar Harbor, Me, USA). Five-month-old B / W mice are randomly assigned to the treatment and control groups. Treatment groups receive either continuous low-dose micellar paclitaxel (2.0 mg / kg; 3 times per week, total of 10 injections) or high-dose “pulse” micellar paclitaxel (20 mg / kg; 4 times, one once a week). The control group receives control micelles.

At predetermined time intervals, paclitaxel-treated B / W mice and untreated controls of comparable age are sacrificed, their spleens are aseptically removed, and single cell suspensions are prepared for lymphocyte counts. To identify spleen lymphocyte subpopulations, fluorescence analysis is performed. The number of cells / million spleen B cells that spontaneously secrete immunoglobulin (IgG, IgM, total immunoglobulin) or anti-ssDNA antibody is determined using an ELISA assay.

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From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications can be made without departing from the scope of the invention. Consequently, the invention is not limited other than by the appended claims.