Pharmaceutical preparation comprising supernatant of blood mononuclear cell culture

Abstract

The present invention relates to a topical pharmaceutical preparation for treating an inflammatory skin condition, preferably a condition associated with ischemia, comprising a supernatant of a physiological solution obtainable by cultivating peripheral blood mononuclear cells (PBMCs) or a subset thereof in a physiological solution free of PBMC-proliferating and PBMC-activating substances for at least 1 h.

Claims

1. A method of topically treating an ischemia associated inflammatory skin condition by a step of administering a topical pharmaceutical preparation to an affected area, wherein the topical pharmaceutical preparation comprises a culture supernatant obtained by in vitro cultivation of peripheral blood mononuclear cells (PBMCs) comprising T cells, B cells, NK cells, and monocytes obtained by Ficoll density gradient centrifugation, wherein the PBMCs are cultivated for at least 1 h within a physiological solution that is a culture medium free of phytohemagglutinin (PHA) and lipopolysaccharide (LPS), wherein before or during the course of cultivation, the PBMCs are subjected to a stress inducing condition, that includes irradiation with at least 60 Gy, and wherein the culture supernatant includes a secretome produced by said PBMCs during in vitro cultivation within said physiological solution following exposure to the stress inducing condition.

2. The method of claim 1, wherein the physiological solution is a physiological salt solution.

3. The method of claim 1, wherein the culture medium includes whole blood or a blood fraction.

4. The method of claim 3, wherein the blood fraction is serum.

5. The method of claim 1, wherein the PBMCs are cultivated following said stress inducing conditions for a period of at least 4 h.

6. The method of claim 5, wherein said period is at least 6 h.

7. The method of claim 6, wherein said period is at least 12 h.

8. The method of claim 1, wherein said topical pharmaceutical preparation is provided as a gel, ointment, dermal patch, cream, powder, liniment, or lotion.

9. The method of claim 8, wherein said topical pharmaceutical preparation is a dermal patch comprising a pharmaceutically acceptable matrix.

10. The method of claim 9, wherein said pharmaceutically acceptable matrix comprises a collagen/elastin matrix.

11. The method of claim 1, wherein the culture supernatant is lyophilized.

12. The method of claim 1, wherein said ischemia associated skin condition is selected from the group consisting of wounds, chronic wounds, diabetic wounds, skin ulcer, skin burns, skin flaps in plastic surgery, and tissue regeneration after dental grafting.

13. A method of topically treating an ischemia associated inflammatory skin condition by a step of administering a topical pharmaceutical preparation comprising a culture supernatant including components from a secretome produced in vitro by peripheral blood mononuclear cells (PBMCs) comprising T cells, B cells, NK cells, and monocytes separated from whole blood by Ficoll density gradient centrifugation, wherein the culture supernatant including components from the secretome is obtained by in vitro cultivation of the PBMCs for at least 1 h within a physiological solution that is a culture medium free of phytohemagglutinin (PHA) and lipopolysaccharide (LPS), wherein before or during the course of cultivation, the PBMCs are subjected to a stress inducing condition that includes irradiation with at least 60 Gy.

14. A method of topically treating an ischemia associated inflammatory skin condition by a step of administering a topical pharmaceutical preparation comprising a culture supernatant produced in vitro by non-proliferating peripheral blood mononuclear cells (PBMCs) comprising T cells, B cells, NK cells, and monocytes obtained by Ficoll density gradient centrifugation, wherein the culture supernatant includes a non cell-surface moiety triggered secretome production triggered by in vitro cultivation of the non-proliferating PBMCs for at least 1 h in a physiological solution free of phytohemagglutinin (PHA) and lipopolysaccharide (LPS), wherein before or during the course of cultivation, the PBMCs are subjected to irradiation with at least 60 Gy.

15. The method of claim 14, wherein the secretome in the topical pharmaceutical preparation consists essentially of the secretome produced by a combination of irradiated, non-proliferating and non-activated T cells, B cells, NK cells, and monocytes.

Description

(1) The present invention is further illustrated by the following figures and examples, however, without being restricted thereto.

(2) FIG. 1 shows the effect of peripheral blood mononuclear cell (PBMC)-derived culture supernatants (SN) on cell migration in human primary keratinocytes (KC) and dermal fibroblasts (FB).

(3) KC and FB were grown in KC-growth medium and DMEM (supplemented with 10% FBS), respectively. After reaching confluency the cell monolayer was scratched with a pipette-tip and further cultivated with PBMC-derived SN for 16 h. We could only detect little effect of SN from living PBMCs (LL) on KC, whereas SN from apoptotic PBMCs (APO) strongly induced KC-migration. In contrast both SN (LL and APO) strongly induced cell migration in dermal FB.

(4) FIG. 2 shows the effect of PBMC-derived SN on the cell cycle progression of KC and FB.

(5) Proliferating KC and FB were cultivated in PMBC-derived SN. After 24 h cells were incubated with BrdU for 2 h and further treated as indicated in the user manual (BrdU-FACS flow cell cycle kit, BD Biosciences). As shown in FIG. 2, stimulation of FB with PMBC-derived SN led to a decrease of the proliferating cell population accompanied by an increase of cells in the G2/M phase. In contrast, a significant increase of proliferating KC with both LL- and APO-SN was found. However, the effect of SN derived from apoptotic cells was more pronounced in KC.

(6) FIG. 3 shows PBMC-derived SN strongly enhance wound-healing in vivo.

(7) Creams containing either LL (FIG. 3a, b) or APO (FIG. 3b) PBMC-SN were applied on 6 mm punch biopsy wounds on the backs of B6/129 mice immediately after wounding. 8 days after wounding the mice were sacrificed and the wounds were analyzed by H&E-staining. As shown in FIGS. 3a and 3b both SN strongly enhanced wound healing in both the dermal and epidermal compartments of the skin.

(8) FIG. 4 shows PBMC-derived SN strongly enhance wound-healing in vivo.

(9) The wound-size during the first 5 days after wounding until a crust was formed was measured. As shown in FIG. 4 it was found that wound-closure after treatment with creams containing PBMC-derived supernatants was much faster compared to the cream alone during the whole 5 days. Whereas the wound-size increased somewhat during the first 2 days with cream alone, wounds treated with PBMC-derived supernatants began to close during the first 24 h after wounding and application of the creams.

(10) FIG. 5 shows increased angiogenesis in mouse wounds in vivo after treatment with PBMC-derived SN.

(11) By immuno-histochemistry for factor VIII (FIG. 5a) and CD31 (FIG. 5b), both markers for blood-vessels, a massive increase in CD31 positive cells in SN treated wounds compared to controls, indicative of increased angiogenesis, which could contribute to the enhanced wound-healing, was found. In contrast an increased number of proliferating cell at this time point as analyzed by Ki67 staining could not be detected (FIG. 5a).

(12) FIG. 6a shows that neither unstimulated viable PBMC or IA-PBMC secrete the mainly monocyte derived pro-inflammatory cytokine TNF-α. (Significances are indicated as follows: * p=0.05, ** p=0.001; n=8)

(13) FIG. 6b demonstrates a strong induction of pro-inflammatory Interferon-γ secretion after activation as compared to unstimulated PBMC. (Significances are indicated as follows: * p=0.05, ** p=0.001; n=8)

(14) FIG. 7a shows pooled results of flow cytometric analysis. PBMCs were gated for T cells and expression of activation markers CD69 and CD25 were evaluated. (Significances are indicated as follows: * p=0.05, ** p=0.001; n=4)

(15) FIG. 7b displays a representative FACS analysis of PBMCs either activated (PHA, CD3 mAb). Gating represents % of positive cells.

(16) FIG. 8 shows high proliferation rates as measured by 3[H]-thymidine incorporation of stimulated PBMC when compared to viable PBMC cultured in RPMI without stimulation.

(17) FIG. 9 shows inhibition of T cell response of PBMC secretoma in T cell proliferation assays.

(18) FIG. 10 shows anti-CD3 and PHA stimulation experiments performed with PBMC.

(19) FIG. 11 shows the proliferation of PBMC upon stimulation with anti-CD3, PHA and mixed lymphocytes.

(20) FIG. 12 shows the level of Annexin V and PI positivity of the supernatant of CD4+ cells inoculated with PBMC supernatants.

(21) FIG. 13 shows the inhibition of the up-regulation of CD25 and CD69 in CD4+ cells by PBMC supernatant.

(22) FIG. 14 shows that the demonetizing of IL-10 and TGF-β did not increase she proliferation rates of CD4+ cells.

EXAMPLES

Example 1

Culture Supernatants of Peripheral Blood Mononuclear Cells Strongly Enhance Wound Healing

(23) Non-healing skin ulcers are often resistant to most common treatments. In a previous study it was shown that application of peripheral blood mononuclear cells (PBMCs) together with basic fibroblast growth factor appeared to be a useful treatment for diabetic gangrene. In the present example it was investigated whether culture supernatants of PBMCs (either non-irradiated or irradiated) are sufficient to induce enhanced wound healing in a mouse model. Furthermore the effect of these supernatants on human primary fibroblasts (FB), keratinocytes (KC) and endothelial cells (EC) was analyzed.

(24) By incubation of FE and KC with PBMC-derived supernatants it was found that supernatants of both non-irradiated and irradiated cells strongly induced migration of FB, whereas they had no effect on FB-proliferation. By contrast, it was shown that both supernatants were effective on KC with respect to their migration- and proliferation capacity. However the effect of supernatants derived from irradiated cells was more pronounced. Since PBMC-derived supernatants induced the migratory and proliferatory machinery in vitro, it was further investigated whether these supernatants are also able to induce wound-healing in vivo. Therefore PBMC-supernatant containing creams were prepared and applied on 6 mm punch biopsy wounds on the backs of B6/129 mice immediately after wounding. The wound-size was measured during the next 4-5 days until a crust was formed. It was surprisingly found that wound-closure after treatment with creams containing PBMC-derived supernatants was much faster compared to the cream alone during the whole 5 days. Interestingly, whereas the wound-size increased somewhat during the first 2 days with cream alone, wounds treated with PBMC-derived supernatants began to close during the first 24 h after wounding and application of the creams. 8-10 days after wounding the mice were sacrificed and the wounds were analyzed by H&E-staining and by immuno-histochemistry for CD31, a marker for blood-vessels. H&E staining revealed that wound healing in both the dermal and epidermal compartments of the skin was more advanced in the presence of creams from PBMC-derived supernatants. Furthermore there was a massive increase in CD31 positive cells in such wounds, indicative of increased angiogenesis, which could contribute to the enhanced wound-healing.

(25) In summary it was shown that PBMC-derived supernatants led to enhanced wound-healing in mice in vivo and that these supernatants also induced proliferation and migration in human cells in vitro. The formulation of creams containing PBMC-supernatants might represent a big advantage for the treatment of non-healing skin ulcers (see FIG. 1 to 5).

Example 2

Resting Peripheral Blood Mononuclear Cells (PBMC) Evidence Low Activation Marker and Reduced Inflammatory Cytokine Production

(26) Activated peripheral blood mononuclear cells (PBMCs) and their supernatants (SN) are supposed to be beneficial in wound regeneration (Holzinger C et al. Eur J Vasc Surg. 1994 May; 8(3): 351-6). In examples 1 and 2 it could be shown that non-activated PBMC and SN derived thereof has beneficial effects in an experimental acute myocardical infarct (AMI) and wounding model. Since non-activation of PBMC had to be verified experimentally it was investigated whether cultivation of PBMC leads to enhanced T-cell activation markers (CD69, CD25) or enhanced inflammatory cytokine secretion (monocyte activation=TNFα, T-cell activation=INFγ). In a control experiment cultured T cells were triggered by CD3 mAb stimulation or Phytohemagglutinin (PHA).

(27) Methods and Results

(28) Venous blood was collected in EDTA-tubes from healthy volunteers. After Ficoll-Hypaque density grade separation, PBMC were collected and divided into viable and irradiated apoptotic cells (IA-PBMC). To obtain apoptotic cells, PBMC were irradiated with 60 Gy (Caesium-137). For flow cytometric analysis 500,000 PBMC were cultivated in 200 μl serum-free medium. Cells were either stimulated with PHA (7 μg/mL) or CD3-mAb (10 μg/mL) or were left unstimulated. After 24 h of incubation cells were washed, stained for CD3, CD69 and CD25 (R&D System) and evaluated for surface activation markers on a FC500 (Coulter). For ELISA assays PBMC were cultivated overnight at a density of 2.5×10.sup.6 cells/ml, either with or without PHA or CD3 stimulation. After 24 h supernatants were harvested and frozen at −20° C. Commercially available ELISA kits for TNF-α (R&D) and INF-γ (Bender) were purchased. In short, MaxiSorp plates were coated with anti-bodies against TNF-α and TNF-γ and stored overnight. After 24 h, plates were washed and samples added in duplicates to each well. After incubation and addition of a detection antibody and Strep-Lavidin-HRP, TMB-substrate was added to each well. After color development, the enzymatic reaction was stopped by addition of sulphic acid. Optical density values were read on a Wallac Victor3 plate reader.

(29) Results:

(30) FACS analysis: CD3 and PHA stimulated T cells showed an upregulation of activation markers CD69 and CD25 after 24 h of incubation. Unstimulated and apoptotic cells expressed only low amounts of CD69 and CD25 (FIG. 6a (representative sample, FIG. 6b, histogram, n=4). Statistical significance is indicated by asterix (xx p<0.001, x p<0.05). ELISA analysis: Whereas neither TNF-α and INF-γ in unstimulated PBMC-derived supernatants were detected, supernatants from PHA or CD3 stimulated PBMC evidenced high values for these cytokines as indicated by ELISA analysis (asterix ** p<0.001, * p<0.05, n=8). The results clearly show a different secretion pattern of inflammatory cytokines in comparison to unstimulated PBMC.

(31) Conclusion:

(32) These data indicate that “unstimulated PBMC” evidence a distinct different phenotype (activation marker, cytokine secretion) as compared to stimulated PBMCs (PHA and CD3 mAb).

(33) FIG. 6a indicates that neither unstimulated viable PBMC or IA-PBMC secrete the mainly monocyte derived pro-inflammatory cytokine TNF-α. (Significances are indicated as follows: * p=0.05, ** p=0.001; n=8)

(34) FIG. 6h demonstrates a strong induction of pro-inflammatory Interferon-γ secretion after activation as compared to unstimulated PBMC. (Significances are indicated as follows: * p=0.05, ** p=0.001; n=8)

(35) FIG. 7a shows pooled results of flow cytometric analysis. PBMCs were gated for T cells and expression of activation markers CD69 and CD25 were evaluated. (Significances are indicated as follows: * p=0.05, ** p=0.001; n=4)

(36) FIG. 7b displays a representative FACS analysis of PBMCs either activated (PHA, CD3 mAb). Gating represents % of positive cells.

Example 3

Proliferative Activity of PBMC Cultivated in a Physiological Solution

(37) The aim of this example is to prove that PBMC have no proliferative activity as compared to immune assays that utilize specific (CD3), unspecific (lectin, PHA) and allogeneic T-cell triggering (mixed lymphocyte reaction, MLR) in a 2 day (CD3, PHA) and 5 day (MLR) stimulation assay.

(38) Material and Methods

(39) PBMC were separated from young healthy volunteers by Ficoll density gradient centrifugation and resuspended in RPMI (Gibco, USA) containing 0.2% gentamycinsulfate (Sigma Chemical Co, USA), 1% L-Glutamin (Sigma, USA) at 1*10.sup.5 cells per 200 μL. Responder cells were either stimulated by MoAb to CD3 (10 μg/mL, BD, NJ, USA), PHA (7 μL/mL, Sigma Chemical Co, USA) or with irradiated allogeneic PBMC at a 1:1 ratio (for MLR). Plates were incubated for 48 h or 5 days and then pulsed for 18 h with 3[H]-thymidine (3.7*10.sup.4 Bq/well; Amersham Pharmacia Biotech, Sweden). Cells were harvested and 3[H]-thymidine incorporation was measured in a liquid scintillation counter.

(40) Results:

(41) Stimulated PBMC showed high proliferation rates as measured by 3[H]-thymidine incorporation when compared to viable PBMC cultured in RPMI without stimulation (FIG. 8). This effect was observed by adding T cell specific stimuli (PHA, CD3) as well as in assays where proliferation was triggered by antigen presenting cells (MLR).

(42) Conclusion:

(43) This set of experiments implicates that viable PBMC held in culture for up to 5 days do not proliferate whereas PENS stimulated by different ways showed a marked proliferative response. It is concluded that culture of PBMC without stimulation does not lead to proliferative response.

Example 4

Secretoma of Separated PBMC Kept Under Sterile Culture Conditions Possess Neo-Angionetic Capacity

(44) Since neo-angionesis and inflammation are strongly linked in vivo it was investigated whether these secretoma of PBMC also exhibit anti-proliferative effects on T cells and therefore interfere with an inflammatory immune response.

(45) Material and Methods

(46) Secretoma were obtained by incubating PBMC (2.5*10.sup.6/mL) from young healthy volunteers separated by Ficoll density gradient centrifugation for 24 h in RPMI (Gibco, CA, USA) containing gentamycinsulfate (Sigma Chemical Co, USA), 1% L-Glutamin (Sigma, USA). Supernatants were separated from the cellular fraction and stored at −80° C. For proliferation assays allogeneic PBMC were resuspended at 1*10.sup.5 cells per 200 μL RPMI after separation. Responder cells were either stimulated by MoAb to CD3 (10 μg/mL, BD, USA) or PHA (7 μL/mL, Sigma Chemical Co, USA). Different dilutions of supernatants were added. Plates were incubated for 48 h and then pulsed for 18 h with .sup.3[H]-thymidine (3.7*10.sup.4 Bq/well; Amersham Pharmacia Biotech, Sweden). Cells were harvested and .sup.3[H]-thymidine incorporation was measured in a liquid scintillation counter.

(47) Results:

(48) Secretoma of allogeneic PBMC evidenced a significant reduction of proliferation rates measured by .sup.3[H]-thymidine incorporation when compared to positive controls (FIG. 9). This effect was dose-dependent and could be seen upon anti-CD3 as well as upon PHA stimulation.

(49) Implication:

(50) This set of experiments implicates that secretoma obtained from viable PBMC held in culture for 24 h exhibit significant anti-proliferative effects in vitro. These data indicate that supernatant derived from PBMC or in lyophilised form may serve as potential therapeutic formula to treat human diseases that are related to hypoxia induced inflammation or other hyperinflammatory diseases (e.g. auto-immune diseases, inflammatory skin diseases).

Example 5

Paracrine Factors Secreted by Peripheral Blood Mononuclear Cells Posses Immunesuppressive Features

(51) In Example 1 anti-inflammatory effects of PBMC secretoma in an acute myocardial infarction (AMI) animal model are evidenced. In this example it is shown that the application of PBMC secretoma after AMI induction inhibits the inflammatory damage of the heart muscle by massively down-regulating the immune response.

(52) Based on these findings possible immunesuppressive effects of secretoma in vitro experiments were investigated. CD4+ cells play a key role in the orchestration of the immune response as they are pivotal for the assistance of other leukocytes (e.g. macrophages, B cells, cytotoxic T cells) in immunological processes.

(53) Material and Methods

(54) Production of PBMC Secretoma

(55) PBMC from healthy volunteers were separated by Ficoll density centrifugation. Cells were resuspended in Ultra Culture Medium (Lonza, Basel, Switzerland) at a concentration of 1*10.sup.6 cells/mL (sup liv). For the production of secretoma from apoptotic PBMC apoptosis was induced by irradiation with 60 Gy (sup APA). Cells were incubated for 24 h in a humidified athmosphere (5% CO2, 37° C., relative humidity 95%). Supernatants were removed and dialysed with a 3.5 kDa cutoff (Spectrum laboratories, Breda, The Netherlands) against 50 mM ammonium acetate overnight at 4° C. Then supernatants were sterile filtrated and lyophilized. Lyophilized secretoma were stored at −80° C. and freshly resuspended for every experiment. Secretoma were random sampled for their pH value.

(56) Separation of CD4 Cells

(57) CD4+ cells were separated by depletion of non-CD4+ T cells utilizing a MACS bead system (Miltenyi, Bergisch Gladbach, Germany). Cells were freshly prepared and immediately used for each experiment.

(58) Measurement of Apoptosis

(59) Apoptosis was detected by flow cytometry using a commercially available Annexin V/PI kit (BD, New Jersey, USA). Apoptosis were defined by Annexin positive staining, late apoptosis by PI positivity.

(60) Proliferation Experiments

(61) PBMC or purified CD4+ cells were diluted in Ultra Culture supplemented with 0.2% gentamycinsulfate (Sigma, St. Louis, Mo., USA), 0.5% β-mercapto-ethanol (Sigma, St Louis, Mo., USA) and 1% GlutaMAX-I (Invitrogen, Carlsbad, Calif., USA) to a concentration of 1*10.sup.5/well in a 96 round-bottom well plate. Cells were stimulated with either PHA (7 μg/mL, Sigma, USA), CD3 (10 μg/mL, BD, New Jersey, USA) IL-2 (10 U/mL, BD, USA) or an 1:1 ratio of allogeneic irradiated (60 Gy) PBMC for MLR. Cells were incubated for 48 h or 5 days (MLR) with different concentrations of PBMC secretoma, IL-10 or TGF-β. Then cells were pulsed for 18 h with 3[H]-thymidine (3.7×10.sup.4 Bq/well; Amersham Pharmacia Biotech, Uppsala, Sweden). Cells were harvested and 3[H]-thymidine incorporation was measured in a liquid scintillation counter.

(62) Activation Markers

(63) Purified CD4+ cells were stimulated with anti-CD3 (10 μg/mL) and co-incubated with different concentration of PBMC secretoma. Cells were stained for CD69 and CD25 following a standard flow cytometric staining protocol and analyzed on a flow cytometer FC500 (Beckman Coulter, Fullerton, Calif., USA).

(64) Results

(65) In preliminary experiments the anti-proliferative properties of PBMC supernatants from viable cells (sup liv) were tested. In anti-CD3 and PHA stimulation experiments proliferations rates were significantly reduced by the addition of secretoma (n=10).

(66) Based on these findings the effect of PBMC secretoma on the T-helper cell compartment was evaluated, since these cells play a pivotal role in launching and perpetuating an immune response. In analogy to FIG. 10 highly purified CD4+ cells lost their proliferative capacity by the addition of secretoma. This phenomenon was observed for the supernatant of living as well as of apoptotic, irradiated PBMC (FIG. 11, n=5).

(67) The next step was to determine possible effects of the secretoma on cell viability. Therefore resting CD4+ cells were inoculated with supernatant and Annexin V and PI positivity was evaluated. Supernatants from both, living and apoptotic PBMC, evidenced remarkable pro-apoptotic effects (FIG. 12, n=5).

(68) To test if PBMC secretoma were able to inhibit CD4+ cell activation the T cell activation markers CD25 and CD69 following anti-CD3 stimulation of CD4+ cells was evaluated. The up-regulation of both markers was significantly and dose-dependent inhibited by PBMC secretoma (FIG. 13, n=5).

(69) In a last set of experiments the effect of the immune-suppressive cytokines IL-1C and TGF-β by the addition of neutralizing antibodies in these experiments was examined. Neither IL-10 and TGF-(was found to be responsible for the anti-proliferative effects of our PBMC secretoma, since demonetizing these cytokines did not increase proliferation rates (FIG. 14, n=5).

(70) Conclusion

(71) These experiments evidence for the first time that PBMC secretoma posses immune-suppressive features in vitro. It was shown that supernatant a) reduces proliferation rates in anti-CD3, PHA and MLR stimulation experiments, b) has the potency to induce apoptosis and inhibits activation of CD4+ cells upon T cell triggering.