Pharmaceutical composition of oxidised avidin suitable for inhalation

Abstract

The present invention describes oxidized avidin, suitable for inhalation, for conditioning the lung affected by inoperable/diffuse diseases, enabling the targeted delivery of biotinylated therapeutic agents to it.

Claims

1. A method for treating a lung cancer disease comprising: administering to a patient in need thereof an effective amount of oxidized avidin via nebulization and inhalation of a pharmaceutical inhalable formulation comprising oxidized avidin, a sterile buffer solution comprising sodium acetate at pH of from 5.0 to 6.9, and a non-ionic agent selected from the group consisting of mannitol, glycerol, glucose, lactose, trehalose, sucrose, propylene-glycol, sorbitol, xylitol, polyethylene-glycol, ethanol and isopropanol, wherein the oxidized avidin is at a concentration of from 0.005% to about 0.5% (w/v), and thereafter administering to the patient a biotinylated therapeutic agent selected from the group consisting of radioactive agents, monoclonal antibodies, cytokines, chemokines, enzymes, chemotherapeutics, viral or plasmid vectors and cells.

2. The method according to claim 1, wherein the biotinylated therapeutic agent is a biotinylated derivative of a monoclonal antibody selected from the group consisting of anti-EGFR, anti-CEA, anti-MUC1, anti-EpCAM, anti-cMET, anti-CTL4, anti-TNF, anti-Tweak, anti-IL-17, anti-IL-23, anti-IL-6, and anti-IL-1.

3. The method according to claim 1, wherein the biotinylated therapeutic agent is a biotinylated adduct of a cytokine selected from the group consisting of TNF, Tweak, TRAIL, gamma interferon, G-CSF, GM-CSF, IL-2, and IL-12.

4. The method according to claim 1, wherein the biotinylated therapeutic agent is a biotinylated adduct of a chemokine selected from the group consisting of CXC and CC chemokine families.

5. The method according to claim 1, wherein the biotinylated therapeutic agent is biotin-DOTA labelled with a radioisotope selected from the group consisting of .sup.52Fe, .sup.52mMn, .sup.55Co, .sup.64Cu, .sup.67Cu, .sup.67Ga, .sup.68Ga, .sup.99mTc, .sup.111In, .sup.123I, .sup.125I, .sup.131I, .sup.32P, .sup.47Sc, .sup.90Y, .sup.109Pd, .sup.111Ag, .sup.149Pm, .sup.186Re, .sup.188Re, .sup.211At, .sup.212Pb, .sup.212Bi and .sup.177Lu.

6. A kit comprising a pharmaceutical inhalable formulation contained in a nebulizer and comprising oxidized avidin, a sterile buffer solution comprising sodium acetate at pH of from 5.0 to 6.9, and a non-ionic agent selected from the group consisting of mannitol, glycerol, glucose, lactose, trehalose, sucrose, propylene-glycol, sorbitol, xylitol, polyethylene-glycol, ethanol, and isopropanol, wherein the oxidized avidin is at a concentration of from 0.005% to about 0.5% (w/v).

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1:

(2) It shows the tissue residence of the complex oxidized avidin/.sup.111In-ST2210 in the eye (drop deposition), tongue (i.m. injection), limb (i.m. injection), skin (drop deposition), scraped skin (drop deposition).

(3) FIG. 2:

(4) It shows the linkage of intravenously injected 68-Ga-ST2210 to a surgical lesion performed in the bladder of a pig to simulate the removal of a human superficial bladder carcinoma (arrow). The image was obtained 4 hours after 68-Ga-ST2210 injection, by Positron Emission Tomography (PET).

(5) FIG. 3:

(6) It shows the chemical integrity of oxidized avidin after having been nebulised for 1 hour in a 100 mM sodium acetate solution at pH 5.5 and without excipient.

(7) FIG. 4:

(8) It shows, at T=2 hours and T=24 hours, the distribution of intravenously injected .sup.111In-ST2210 in various organs of mice which have received 24 hour before oxidized avidin or vehicle through inhalation.

(9) FIG. 5:

(10) Immunochemistry with mouse anti-avidin antibody of lung sections, 24 hours after oxidized avidin aerosol exposure.

(11) FIG. 6:

(12) PET imaging of .sup.64Cu-ST2210 intravenously injected mice which were exposed to oxidized avidin aerosol 24 hours before.

(13) FIG. 7:

(14) EGFR+ A431 cells incubated with biotinylated cetuximab at the dose of 5 g, 0.05 g, 0.05 ng and 0.05 pg/ml for 1 hour in PBS without (a) or with (b) pre-treatment with oxidized avidin.

(15) FIG. 8:

(16) It shows the inhibition of proliferation of A431 cell line with biotinylated cetuximab with or without prior treatment with oxidized avidin.

(17) FIG. 9:

(18) It show a cytofluorimetry analysis of the induction of apoptosis by biotinylated cetuximab on two cell lines, one expressing high level of EGFR (i.e., A431) and one which does not express EGFR (i.e., SKMe128) with or without treatment with oxidized avidin.

(19) FIG. 10:

(20) It shows the inhibition of proliferation induced by biotinylated cetuximab with or without pre-treatment with AvidinOX on A431, A549 (i.e., lung carcinoma expressing low levels of EGFR, KRAS mutated) and SKMe128 cells.

EXAMPLES

Example 1

(21) Balb/c mice of about 20 g were treated in the indicated sites with a solution of avidin or oxidized avidin as obtained following the procedure described at example 1 of WO2009016031 (3.0 mg/ml dissolved in 100 mM sodium acetate, pH 5.5) pre-complexed with .sup.111In-ST2210. Twenty four hours after injection/deposition, the mice were sacrificed by CO.sub.2 asphyxia and the treated sites analysed by means of a gamma counter. Data are reported in FIG. 1, and are expressed as the % of injected dose/100 mg of tissue (% ID/100 mg). Results show a statistically significant higher amount of oxidized avidin/.sup.111In-ST2210 complex in injected tongue, limb muscle and on the topically treated scraped skin compared to the Avidin/.sup.111In-ST2210 complex. However, deposition of the oxidized avidin/.sup.111In-ST2210 or Avidin/.sup.111In-ST2210 complex on normal skin or in the eye did not lead to similar results, indicating that the complex does not bind to external tissue surfaces and consequently that a surgical operation is required to take advantage of the binding properties of oxidized avidin either as single agent when complexed with a biotinylated agent.

Example 2

(22) Anesthetised female pig of about 40 kg was subjected to surgery to generate two 2 cm superficial lesions on the bladder wall. Then, 30 ml of oxidized avidin solution (3.0 mg/ml dissolved in 100 mM sodium acetate, pH 5.5) were instilled via a catheter and let interact for 1 hour. Bladder was then washed with saline and 0.5 g 68-Ga-ST2210 was i.v. administered. After 4 hours the pig was subjected to PET. Results as shown in FIG. 2, demonstrate that only the region that had been subjected to surgery (i.e., 2 mm lesion) enabled the binding of oxidized avidin, meanwhile intact bladder tissue proved to be completely inert to the aldehyde moieties of oxidized avidin. Those surprising data are however, in agreement with those of example 1 wherein non-surgically damaged tissue were inert to oxidized avidin.

Example 3

(23) A 100 mM sodium acetate solution at pH 5.5 containing oxidized avidin at the concentration of 3.0 mg/ml was nebulised by means of a the Nose-Only inExpose System (Scireq-EMKA technologies) for 1 hour at room temperature. The particle size of the protein solution midst was 5 m. The nebulised solution was recovered in a falcon tube and analyzed by HPLC. Data in FIG. 3 show the same elution profile for the oxidized avidin solution pre and post nebulisation indicating perfect stability of the protein. This result was not obvious as many proteins and nucleic acid need extensive formulation studies to select a condition that preserves integrity and potency of such drugs during nebulisation (Geller D. E., et al., J. Aerosol Med. Pulm. Drug Deliv., 2010, 23 Suppl 1, S55; Markovic S. N., et al., Am. J. Clin. Oncol., 2008, 31, 6, 573; Choi W. S., et al., Proc. Natl. Acad. Sci., 98, 20, 11103).

(24) It was very interesting to note that notwithstanding the fact that aldehyde derivatives are highly susceptible to hydration in the presence of water, no such phenomena was observed during oxidized avidin nebulisation process. Indeed, the number of aldehyde moieties per molecule, as determined by Purpald's method (Quesenberry M. S., et al., Anal. Biochem., 1996, 234, 1, 50), was found to be substantially the same (considering the variability of the assay) before and after nebulisation at both pH 5.0 and pH 5.5, with and without mannitol excipient as demonstrated by data in Table 1.

(25) TABLE-US-00001 TABLE 1 Number of CHO moieties per molecule Before After Oxidized avidin batch nebulisation nebulisation Mannitol formulation pH 5.0 19.7 19.2 Mannitol formulation pH 5.5 17.1 19.4 Acetate buffer formulation pH 5.5 16.4 16.7

Example 4

(26) Biological activity and biostribution of nebulised oxidized avidin were evaluated in rat by measuring the uptake of 5 g of .sup.111Indium radiolabelled biotinDOTA (i.e., .sup.111In-ST2210) intravenously injected, 24 hours after oxidized avidin, avidin (3.0 mg/ml solution, 0.8 ml/min, 1 hour nebulisation) or vehicle inhalation exposure, in the lung and non target organs.

(27) Twenty Sprague Dawley rats were divided in three different groups. Each group received the following treatments: the first group (made of 4 rats) was exposed to nebulised vehicle only (i.e., 100 mM sodium acetate solution at pH 5.5) and 24 hours later the rats received a 5 g i.v. dose of .sup.111In-ST2210 in 0.5 ml of saline. the second group (made of 8 rats) was exposed to nebulised avidin (about 10 mg/kg) in a 100 mM sodium acetate solution at pH 5.5 and 24 hours later the rats received a 5 g i.v.dose of .sup.111In-ST2210 in 0.5 ml of saline. the third group (made of 8 rats) was exposed to nebulised oxidized avidin (about 10 mg/kg) in a 100 mM sodium acetate solution at pH 5.5 and 24 hours later the rats received a 5 g i.v. dose of .sup.111In-ST2210 in 0.5 ml of saline.

(28) All rats were sacrificed 2 hours after the i.v. administration of .sup.111In-ST2210, and samples of blood, spleen, kidney, liver, stomach, brain, trachea, and tissue samples of different portions of lung were collected, weighed and analyzed with the use of a gamma-counter (Perkin Elmer). Data were expressed as the % of injected dose/gram of tissue (% ID/g).

(29) As shown in Table 2, oxidized avidin inhalation enables to increase in a statistically significant fashion .sup.111In-ST2210 concentration in the lung, meanwhile other organs did not show any statistical differences of concentration of .sup.111In-ST2210 compared to avidin or vehicle groups.

(30) TABLE-US-00002 TABLE 2 .sup.111In-ST2210 % ID/g Groups Vehicle Avidin Oxidized avidin Blood 0.011 0.005 0.009 0.005 0.012 0.004 Spleen 0.105 0.159 0.027 0.003 0.032 0.002 Kidney 0.426 0.082 0.378 0.048 0.414 0.033 Liver 0.046 0.009 0.041 0.007 0.051 0.005 Stomach 0.031 0.011 0.067 0.085 0.276 0.246 Brain 0.002 0.001 0.002 0.000 0.002 0.000 Trachea 0.021 0.007 0.022 0.008 0.029 0.007 Lung 0.024 0.004 0.023 0.006 0.054 0.009*** Lung sn 0.024 0.004 0.023 0.006 0.053 0.008*** Lung dx 0.024 0.004 0.022 0.006 0.055 0.009*** (caudal lobe) Lung dx 0.025 0.004 0.023 0.005 0.055 0.011*** (cranial, middle and accessory lobe) ***p < 0.001 one way Anova versus avidin

Example 5

(31) A biodistribution experiment aimed at determining the lung uptake selectivity and stability of .sup.111In-ST2210 was conducted. Balb/c mice of about 20 g (5 mice/group) were exposed to nebulised (i.e., by means of Nose-Only inExpose SystemScireqEMKA technologies) oxidized avidin (3 mg/ml solution) or vehicle (3 ml) for 1 hour; 1 hour exposition corresponding to about a 90 mg/kg dose. Twenty four hours after, all mice received .sup.111In-ST2210 intravenously (i.e., 1 g in 0.2 ml of saline), and after further 2 or 24 hours, the animals were sacrificed by CO.sub.2 asphyxia. The lung and non target organs were collected weighed and counted in a gamma counter. Data were expressed as the % of injected dose/gram of tissue.

(32) Results, as exposed in FIG. 4, demonstrate a specific and significant uptake of .sup.111In-ST2210 only in the lung which had been pre-treated with oxidized avidin.

Example 6

(33) Balb/c mice of about 20 g were exposed to nebulised oxidized avidin according to the protocol described at example 3, and were sacrificed by asphyxia 24 hours after. The lungs were removed and fixed in formalin and paraffin embedded. Serial sections obtained by means of a microtome were processed and incubated with a HRP-conjugated rabbit anti-avidin antibody (GeneTex, USA) and then with DAB substrate.

(34) FIG. 5 shows the presence of oxidized avidin at broncho/epithelial level down to terminal bronchioles. This distribution was found to be homogenous in all lung compartments.

Example 7

(35) Balb/c mice of about 20 g were exposed to nebulised oxidized avidin or vehicle according to the protocol described at example 5. After 24 hours, they received an intravenous 1 g dose of .sup.64Cu-ST2210. .sup.64Cu-ST2210 distribution was examined by PET imaging 4 hours after. FIG. 6 shows the presence of a radioactive signal in the lungs of the mouse pre-treated with nebulised oxidized avidin but not in the lungs of a vehicle treated mouse. Kidneys, bladder are visible in both groups of mice. This observation is consistent with the physiological elimination of ST2210 at that time point. A radioactive signal in one limb is also visible in both groups of mice since they had been i.m. pre-treated with oxidized avidin at the time of aerosol in order to have an internal positive control in the experiment.

Example 8

(36) Human EGFR.sup.+, epidermoid carcinoma cells A431 were incubated in PBS for 1 hour with 1 ml of an anti-EGFR monoclonal biotinylated antibody (i.e., biotinylated cetuximab) in the dose range of 0.05 pg/ml to 5 g/ml. In one experiment the cells were previously incubated with oxidized avidin meanwhile in the first experiment, cells were only treated with biotinylated cetuximab.

(37) After washings, biotinylated cetuximab binding was detected by cytofluorimetry after incubation with a mouse anti-human antibody conjugated with phycoerythrin (PE). As demonstrated in FIG. 7, in the presence of oxidized avidin, the binding to A431 cells of low doses of biotinylated cetuximab such as 0.05 ng and 0.05 pg/ml is still visible.

Example 9

(38) Data reported in FIG. 8 show that the anti proliferative activity of biotinylated cetuximab (experiment being conducted as reported in example 8) was increased at least 3 times owing to its immobilization through binding to the membrane bound oxidized avidin even at doses as low as 0.05 ng and 0.05 pg/ml. Proliferation inhibition was measured by Cell Titer Glow assay, Promega. Data are expressed as % of inhibition of cell proliferation in the absence of oxidized avidin (i.e., white bars) or in its presence (i.e., black bars). Moreover, as also previously reported (Petronzelli F., et al., Basic Clin. Pharmacol. Toxicol., 2011, 233), oxidized avidin did not affect cell proliferation as observed in a comparison experiment involving medium only (data not shown).

Example 10

(39) Induction of apoptosis by biotinylated cetuximab with or without pre-treatment with AvidinOX was tested on EGFR.sup.+ A431 cells (vulvar squamous cell carcinoma, expressing high levels of EGFR, KRAS wild-type) and EGFR.sup. SKMel28 cells (which do not express EGFR). The cells were incubated for 15 minutes with biotinylated cetuximab (b-cetuximab) in the presence or absence of AvidinOX pre-treatment. After washings, the cells were incubated in complete medium for 18 hours. Annexin V positive cells were analysed by cytofluorimetry with FITC-Annexin V apoptosis detection kit I (BD Pharmingen). Data in FIG. 9 indicate that the pro-apoptotic effect of biotinylated cetuximab is increased at least 3 times when AvidinOX is anchored on the membrane of the EGFR.sup.+ cells but not of the EGFR.sup. cells thus indicating the specificity of pro-apoptotic activity of the AvidinOX-anchored biotinylated cetuximab.

Example 11

(40) Inhibition of proliferation by biotinylated cetuximab with or without pre-treatment with AvidinOX was tested on A431, A549 (i.e., lung carcinoma, low EGFR, KRAS mutated) and SKMel28 cells. For testing the effect of anchored biotinylated cetuximab on cell proliferation, 510.sup.5 A431, A549 and SKMel28 cells, with and without pre-incubation with AvidinOX, were seeded in 96 wells microtiter plates (210.sup.3 cells/well) in DMEM, 10% FCS. After adhesion, 100 l of biotinylated cetuximab were added, in triplicates, in the range of 0.05 pg/ml to 5 g/ml, in DMEM 1% FCS. The cells were washed after 15 minutes and cultivated for 48 hours in DMEM 1% FCS. Cell viability was detected by CellTiter-Glow (Promega). Data in FIG. 10 show that biotinylated cetuximab was able to inhibit proliferation of cells expressing high levels of EGFR (i.e., human vulvar carcinoma A431 cells) and low levels of EGFR (i.e., KRAS mutated, human lung adenocarcinoma A549 cells) but not proliferation of EGFR.sup. cells (SKMel28). Surprisingly, such effects were significantly improved when biotinylated cetuximab was AvidinOX-anchored on the cell surface of these EGFR.sup.+ cells.