DRY POWDER COMPOSITION COMPRISING LONG-CHAIN RNA

Abstract

The present invention is directed to a storage-stable formulation of long-chain RNA. In particular, the invention concerns a dry powder composition comprising a long-chain RNA molecule. The present invention is furthermore directed to methods for preparing a dry powder composition comprising a long-chain RNA molecule by spray-freeze drying. The invention further concerns the use of such a dry powder composition comprising a long-chain RNA molecule in the preparation of pharmaceutical compositions and vaccines, to a method of treating or preventing a disorder or a disease, to first and second medical uses of such a dry powder composition comprising a long-chain RNA molecule and to kits, particularly to kits of parts, comprising such a dry powder composition comprising a long-chain RNA molecule.

Claims

1. Dry powder composition comprising a long-chain RNA molecule.

2. The dry powder composition according to claim 1, which comprises a plurality of particles.

3. The dry powder composition according to claim 1 or 2 having a residual moisture content of 7% (w/w) or less.

4. The dry powder composition according to any one of claims 1 to 3, wherein the median particle size in a volume weighted distribution is at least 1 μm.

5. The dry powder composition according to any one of claims 1 to 4, wherein the average sphericity of the particles is in a range from 0.7 to 1.0.

6. The dry powder composition according to any one of claims 1 to 5, wherein the long-chain RNA molecule is present in the form of a free long-chain RNA molecule, or in the form of a complex comprising the long-chain RNA molecule.

7. The dry powder composition according to any one of claims 1 to 6, wherein the long-chain RNA molecule is in a complex with a cationic or polycationic compound.

8. The dry powder composition according to any one of claims 1 to 7, wherein the long-chain RNA molecule is a single-stranded RNA molecule.

9. The dry powder composition according to any one of claims 1 to 8, wherein the long-chain RNA molecule comprises at least 30 nucleotides.

10. The dry powder composition according to any one of claims 1 to 9, wherein the long-chain RNA molecule comprises more than 200 nucleotides, preferably at least 250 nucleotides.

11. The dry powder composition according to any one of claims 1 to 10, wherein the long-chain RNA molecule comprises at least one open reading frame (ORF) encoding a protein or a peptide.

12. The dry powder composition according to any one of claims 1 to 11, wherein the long-chain RNA molecule is an mRNA molecule.

13. The dry powder composition according to any one of claims 1 to 12, wherein the long-chain RNA molecule comprises at least one modification.

14. The dry powder composition according to any one of claims 1 to 13, which comprises at least one further excipient.

15. Method for preparing a dry powder comprising a long-chain RNA molecule, wherein the method comprises the following steps: a) providing a liquid comprising the long-chain RNA molecule, b) drying the liquid provided in step a) by spray-freeze drying.

16. The method according to claim 15, wherein the long-chain RNA molecule is characterized by any one of the features defined in any one of claims 6 to 13.

17. The method according to claim 15 or 16, wherein the liquid comprising the long-chain RNA molecule further comprises at least one excipient selected from a cryoprotectant, a lyoprotectant and a bulking agent.

18. The method according to any one of claims 15 to 17, wherein the liquid comprising the long-chain RNA molecule does not contain a lipid compound.

19. The method according to any one of claims 15 to 18, wherein the liquid comprising the long-chain RNA molecule comprises a solvent suitable for spray-freeze drying.

20. The method according to any one of claims 15 to 19, wherein the drying comprises a step of spray-freezing and a step of lyophilization.

21. The method according to any one of claims 15 to 20, wherein the liquid comprising the long-chain RNA molecule is atomized and the droplets resulting from the atomization of the liquid are characterized by a mass median aerodynamic diameter of 300 nm to 200 μm.

22. The method according to any one of claims 15 to 21, wherein the liquid comprising the long-chain RNA molecule is atomized by using an atomizer selected from the group of rotary atomizers, pressure nozzles, two-fluid nozzles, ultrasonic nebulizers and vibrating orifice aerosol generators.

23. Dry powder composition obtainable by the method according to any one of claims 15 to 22.

24. Pharmaceutical composition comprising or consisting of the dry powder composition according to any one of claims 1 to 14 or the dry powder composition according to claim 23.

25. The pharmaceutical composition according to claim 24, which comprises at least one further pharmaceutically acceptable excipient.

26. Vaccine comprising or consisting of the dry powder composition according to any one of claims 1 to 14 or the dry powder composition according to claim 23.

27. The vaccine according to claim 26, which comprises at least one further pharmaceutically acceptable excipient.

28. The vaccine according to claim 26 or 27, wherein the dry powder composition was reconstituted in a suitable solvent or buffer.

29. Kit comprising the dry powder composition according to any one of claims 1 to 14 or 23, the pharmaceutical composition according to claim 24 or 25, or the vaccine according to any one of claims 26 to 28, a solvent or buffer for resuspending the dry powder composition, the pharmaceutical composition or the vaccine, and optionally technical instructions comprising information regarding the administration and/or dosage of the dry powder composition, the pharmaceutical composition or the vaccine.

30. Kit of parts comprising in one or more parts of kit the dry powder composition according to any one of claims 1 to 14 or 23, the pharmaceutical composition according to claim 24 or 25, or the vaccine according to any one of claims 26 to 28, a solvent or buffer for resuspending the dry powder composition, the pharmaceutical composition or the vaccine, and optionally technical instructions comprising information regarding the administration and/or dosage of the dry powder composition, the pharmaceutical composition or the vaccine.

31. Use of the dry powder composition according to any one of claims 1 to 14 or 23 for the preparation of a medicament for the prophylaxis, treatment and/or amelioration of a disorder or a disease.

32. The use according to claim 31, wherein the disorder or disease is selected from the group consisting of cancer or tumor diseases, infectious diseases, preferably viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, cardiovascular diseases and neuronal diseases.

33. Method of treating or preventing a disorder or a disease by administering to a subject in need thereof a pharmaceutically effective amount of the dry powder composition according to any one of claims 1 to 14 or 23, the pharmaceutical composition according to claim 24 or 25, or the vaccine according to any one of claims 26 to 28.

34. The method according to claim 33, wherein the disorder or disease is selected from the group consisting of cancer or tumor diseases, infectious diseases, preferably viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, cardiovascular diseases and neuronal diseases.

35. The dry powder composition according to any one of claims 1 to 14 or 23, the pharmaceutical composition according to claim 24 or 25, the vaccine according to any one of claims 26 to 28, the kit according to claim 29 or the kit of parts according to claim 30, for use in the prophylaxis, treatment and/or amelioration of a disorder or disease.

36. The dry powder composition according to any one of claims 1 to 14 or 23, the pharmaceutical composition according to claim 24 or 25, the vaccine according to any one of claims 26 to 28, the kit according to claim 29 or the kit of parts according to claim 30, for use according to claim 35, wherein the disorder or disease is selected from the group consisting of cancer or tumor diseases, infectious diseases, preferably viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, cardiovascular diseases and neuronal diseases.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0234] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

[0235] FIG. 1: Schematic diagram of a spray-freeze drying apparatus [0236] A: Spray-freeze drying process, where liquid feed is atomized and frozen in a cooling zone. B: Frozen particles are subsequently freeze-dried in a lyophilizer.

[0237] FIG. 2: Scheme of a continuous spray-freeze drying process line showing the three process compartments (A: droplet formation through nozzle; cooling and fast freezing of droplets to particles; B: continuous freeze-drying drum; C: flexible multi or single-dose packaging of bulk powder).

[0238] FIG. 3: Sequence of the mRNA used in this study (R2564; SEQ ID NO: 1).

[0239] FIG. 4: Temperature and pressure profile of the lyophilization process in Example 4.

[0240] FIG. 5: Photograph of spray-freeze dried powder of protamine-formulated RNA (T-SFD1, T-SFD2) and spray-freeze dried powder of placebo sample (T-SD-P).

[0241] FIG. 6: Scanning electron microscope (SEM) images of protamine-formulated RNA powder particles (T-SFD-1, T-SFD-2) and placebo powder particles (T-SFD-P).

[0242] FIG. 7: Particle size distribution of spray-freeze dried powders of protamine-formulated RNA (T-SFD-1, T-SFD-2) as measured by laser diffraction (Example 6).

[0243] FIG. 8: X-ray powder diffraction analysis of spray-freeze dried protamine-formulated RNA (T-SFD1, T-SFD2) and of spray-freeze dried placebo sample (T-SFD-P), respectively (Example 8).

[0244] FIG. 9: Particle size distribution of spray-freeze dried powders of protamine-formulated RNA (T-SFD-1, T-SFD-2) as determined by nanoparticle tracking analysis (NTA; Example 10).

[0245] FIG. 10: Particle size distribution of protamine-formulated RNA before (T0) and after (T-SFD 1, T-SFD 2) spray-freeze drying, respectively, and particle size of placebo sample before (T0-P) and after (T-SFD-P) spray-freeze drying. The particle size was determined by dynamic light scattering (DLS; Example 11).

EXAMPLES

[0246] The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

Example 1: Preparation of DNA and RNA Constructs

[0247] A vector for in vitro transcription was constructed containing a T7 promoter followed by a GC-enriched sequence encoding the hemagglutinin (HA) protein of influenza A virus (A/Netherlands/602/2009(H1N1)) and used for subsequent in vitro transcription reactions.

[0248] According to a first preparation, the DNA sequence coding for the above mentioned mRNA was prepared. The constructs R2564 (SEQ ID NO: 1) was prepared by introducing a 5′-TOP-UTR derived from the ribosomal protein 32L4, modifying the wild type coding sequence by introducing a GC-optimized sequence for stabilization, followed by a stabilizing sequence derived from the albumin-3′-UTR, a stretch of 64 adenosines (poly(A)-sequence), a stretch of 30 cytosines (poly(C)-sequence), and a histone stem loop. In SEQ ID NO: 1 (see FIG. 4) and the sequence of the corresponding mRNA is shown.

Example 2: In Vitro Transcription and Purification of RNA

[0249] The respective DNA plasmids prepared according to section 1 above were transcribed in vitro using T7 polymerase. The in vitro transcription of influenza HA encoding R2564 was performed in the presence of a CAP analog (m7GpppG). Subsequently the RNA was purified using PureMessenger® (CureVac, Tübingen, Germany; WO2008/077592A1).

Example 3: Preparation of Protamine-Formulated RNA

[0250] RNA was diluted (0.87 g/L RNA final concentration) and a protamine/trehalose mixture was prepared (43000 anti-heparin IU/L protamine; 10.87% trehalose in water for injection). One volume unit of each solution was mixed to yield a ratio of protamine to RNA of 50 anti-heparin IU per mg RNA.

[0251] The solution of RNA/protamine complexes were supplemented with R2564 to yield final concentrations of 0.4 g/L RNA complexed with 20000 anti-heparin IU/L of protamine (corresponding to a protamine concentration of about 0.15 g/L), 0.4 g/L free RNA and 5% trehalose (w/w).

[0252] Such formulated RNA was used for spray-freeze-drying experiments.

[0253] As a placebo, 5% trehalose was prepared in water for injection.

Example 4: Spray-Freeze Drying of Protamine-Formulated RNA and Placebo Formulation

[0254] The spray-freeze-drying experiments were carried out using the protamine-formulated RNA prepared according to Example 3 or the placebo formulation according to Example 3. Two aliquots (40 ml each) of protamine-formulated RNA (T-SFD1 and T-SFD2) and one aliquot (40 ml) of placebo sample (T-SFD-P) were thawed and each aliquot was homogenized by gentle mixing using a magnetic stirrer before spray-freeze-drying. Spray-freeze-drying was performed in a technical environment. Spray-freezing was carried out by using a PipeJet dispenser. The spray-freezing parameters are summarized in Table 1.

TABLE-US-00001 TABLE 1 Process parameters for spray-freezing Process parameter T-SFD1 T-SFD2 T-SFD-P Nozzle type PipeJet PipeJet PipeJet Pipe diameter [μm] 500 500 500 Number of pipes 3 3 3 Stroke length [μm] 36 28 36 Downstroke speed 500 500 500 [μm/ms] Hold time [μs] 20 20 20 Upstroke speed [μm/ms] 15 15 15

[0255] Approximately 35 ml of each aliquot were spray-frozen. The obtained frozen pellets were transferred into weighed 20R vials (5 vials were filled for each experiment) and stored at −125° C. until lyophilization.

[0256] For lyophilization, the vials containing the frozen pellets were loaded onto an Epsilon 2-12D pilot freeze-dryer. The process parameters for lyophilization are summarized in Table 2. The diagram in FIG. 4 shows the temperatures and pressures as determined over time.

TABLE-US-00002 TABLE 2 Process parameters for lyophilization time temperature pressure total time # step [hh:mm] [° C.] [mbar] [h] 1 loading 00:00 −40 1000 0.0 2 freezing 02:30 −40 1000 2.5 3 primary drying 00:30 −40 0.1 3.0 4 primary drying 00:45 5 0.1 3.8 5 primary drying 10:00 5 0.1 13.8 6 primary drying 05:00 0 0.1 18.8 7 primary drying 15:00 0 0.1 33.8 8 secondary drying 01:00 30 0.1 34.8 9 secondary drying 08:00 30 0.1 42.8

[0257] As a product of the spray-freeze-drying process, a free-flowing white powder was obtained for the protamine-formulated RNA as well as for the placebo formulation (see FIG. 5). The respective yield was calculated and is indicated in Table 3.

TABLE-US-00003 TABLE 3 Yields of the spray-freeze-drying experiments Yield T-SFD1 T-SFD2 T-SFD-P Yield [g] 1.255 1.345 1.231 Theoretical yield* [%] 72 77 70

Example 5: Scanning Electron Microscopy (SEM) of Spray-Freeze-Dried Powder Particles

[0258] Images of spray-dried powder particles were generated by using the bench top scanning electron microscope Phenom (Phenom-World B.V., Eindhoven, Netherlands). The instrument is equipped with a CCD camera and a diaphragm vacuum pump. Each sample was prepared in a glove box under controlled humidity conditions (<20% relative humidity) by using the following method: a small amount of the powder was carefully put on a self-adhesive carbon foil placed on a sample holder. The sample was analyzed under vacuum with a light optical magnification of 24× and 5 kV acceleration voltage. The electron optical magnification was adjusted between 1160× and 1700× and images were made from representative sections of each sample.

[0259] The obtained images (see FIG. 6) demonstrate that the obtained powder particles have spherical shape. The size of the spray-freeze-dried powder particles was in the range from about 100 μm to about 200 μm.

Example 6: Laser Diffraction Analysis of Spray-Freeze-Dried Formulations

[0260] Size distribution of spray-freeze dried powders were measured by laser diffraction. Laser diffraction measurements were performed using a Partica LA-950 Laser Diffraction Particle Size Distribution Analyzer (Horiba, Kyoto, Japan) equipped with a 605 nm laser diode for detecting particles >500 nm and 405 nm blue light emitting diode (LED) for detecting particles <500 nm. The powder samples were dispersed in Miglyol 812 by ultra sonication for up to 5 min. Prior to measurement, the system was blanked with Miglyol 812. Each sample dispersion was measured 3 times. Measurement results were analyzed using Horiba LA-950 Software.

[0261] The results were reported as

[0262] D10: particle diameter corresponding to 10% of the cumulative undersize distribution;

[0263] D50: particle diameter corresponding to 50% of the cumulative undersize distribution;

[0264] D90: particle diameter corresponding to 90% of the cumulative undersize distribution.

[0265] The results are summarized in Table 4 and FIG. 7.

TABLE-US-00004 TABLE 4 Laser diffraction analysis of spray-freeze-dried formulations Mean diameter D10 size D50 size D90 size Sample [μm] [μm] [μm] [μm] T-SFD1 190 122 199 285 T-SFD2 155 82 166 262

Example 7: Residual Moisture Content of Spray-Freeze-Dried Formulations

[0266] The residual moisture content of the dried powders were determined using the coulometric Karl Fischer titrator Aqua 40.00 (Analytik Jena GmbH, Jena, Germany), which is equipped with a headspace module.

[0267] As a system suitability check, the residual moisture content of a Pure Water Standard (Apura 1 water standard oven 1.0, Merck KGaA) was analyzed prior to sample measurement. The residual moisture content of the standard had to be within 1.00±0.03% in order to comply with the manufacturer specifications.

[0268] For the measurement, about 20 mg of sample were weighed into 2R glass vials and heated to a measurement temperature of 120° C. in the oven connected to the reaction vessel via a tubing system. The evaporated water was transferred into the titration solution and the amount of water was determined. The measurement was performed until no more water evaporation was detectable (actual drift comparable to drift at the beginning of the measurement). Ambient moisture was determined by measurement of three blanks (empty vials prepared in the preparation environment). Results obtained for samples were corrected for the determined ambient moisture by blank subtraction. Samples were measured in duplicates. The results are shown in Table 5.

TABLE-US-00005 TABLE 5 Residual water content of spray-freeze-dried formulations Sample Water content [%] T-SFD1 0.21 T-SFD2 0.23 T-SFD-P 0.35

[0269] These results show that spray-freeze-drying can be used in order to obtain dry powder formulation with an extremely low water content. The residual water content of all spray-freeze-dried formulations was further reduced in comparison to the residual water content of lyophilized cakes (<0.6%).

Example 8: X-Ray Powder Diffraction (XRD) Analysis of Spray-Freeze-Dried Formulations

[0270] Wide angle X-ray powder diffraction (XRD) was used to study the morphology of the dried products. The X-ray diffractometer Empyrean (Panalytical, Almelo, The Netherlands) equipped with a copper anode (45 kV, 40 mA, Kα1 emission at a wavelength of 0.154 nm) and a PIXcel3D detector was used. Approximately 100 mg of the spray-freeze dried samples were analyzed in reflection mode in the angular range from 5−45° 2θ, with a step size of 0.04° 2θ and a counting time of 100 seconds per step. The respective diagrams are shown in FIG. 8. It was found that all samples showed an amorphous pattern and no indication of crystalline phases.

Example 9: Reconstitution Behaviour of Spray-Freeze-Dried Formulations

[0271] For reconstitution of the spray-freeze dried samples, the reconstitution volume was calculated for each sample individually based on the amount of powder weighed into the vial. The calculation was based on the method for reconstitution of lyophilized samples (addition of 600 μl water for injection to 30.6 mg powder per vial).

[0272] The reconstitution volume for varying amounts of spray-freeze dried powder was calculated according to the following equation:

V.sub.reconst.=m.sub.powder*1000 μl/51 mg

[0273] V.sub.reconst.: reconstitution volume in ml

[0274] m.sub.powder: mass of powder to be reconstituted in mg

(based on a theoretical solid content of 51 mg per ml (50 mg/ml trehalose, 0.8 mg/ml RNA (free+complexed), 20 anti-heparin IU/mL protamine))

[0275] The spray-freeze dried samples were reconstituted under laminar flow conditions using a procedure comparable to the procedure for lyophilized product: cap and stopper were removed from the vial and the calculated volume of water for injection was added to the dry powder (into the center of the vial) by using a multipette with 10 ml combitip. The vial was carefully slewed (shaking was avoided), until all powder particles were dissolved. The reconstitution time was measured as the time required in order to achieve full reconstitution of the dry powder after the liquid has been added. The reconstitution behavior was judged, mainly with respect to foaming, and recorded (see Table 6). All samples were fully reconstituted in less than one minute and no foaming was observed.

TABLE-US-00006 TABLE 6 Reconstitution behaviour of spray-freeze-dried formulations Sample (~240 mg) Reconstitution time [mm:ss] Foam formation T-SFD1 00:56 0 T-SFD2 00:45 0 T-SFD-P 00:46 0

Example 10: Nanoparticle Tracking Analysis (NTA) of Spray-Freeze-Dried Formulations

[0276] NTA experiments were carried out with a NanoSight LM20 (NanoSight, Amesbury, UK). The instrument is equipped with a 405 nm blue laser, a sample chamber and a Viton fluoroelastomer 0-Ring. The samples were diluted with ultra-pure water in order to achieve suitable concentrations for NTA measurement. After the measurement, all results were normalized to the original concentration.

[0277] Samples were loaded into the measurement cell using a 1 ml syringe. Movements of the particles in the samples were recorded as videos for 60 seconds at room temperature using the NTA 2.0 Software. The recorded videos were analyzed with the NTA 2.0 Software. The results of the NTA analysis are shown in Table 7 and FIG. 9. Spray-freeze-drying resulted in comparable or slightly decreased particle sizes as compared to a lyophilized control (TO).

TABLE-US-00007 TABLE 7 Nanoparticle tracking analysis of spray-freeze-dried formulations Mean Mode D10 D50 D90 size size size size size Total conc. Sample [nm] [nm] [nm] [nm] [nm] [#/ml] T0 124 ± 107 ± 80 ± 118 ± 171 ± 39 8.31 (±0.34) 25 16 12 24 E+11 T-SFD1 108 ± 3  97 ± 4 73 ± 1 103 ± 1 147 ± 10 6.71 (±1.15) E+11 T-SFD2 108 ± 6  94 ± 7 72 ± 2 102 ± 5 148 ± 11 8.69 (±1.27) E+11 T0-P n/a.sup.(1) n/a.sup.(1) n/a.sup.(1) n/a.sup.(1) n/a.sup.(1) n/a.sup.(1) T-SFD-P n/a.sup.(1) n/a.sup.(1) n/a.sup.(1) n/a.sup.(1) n/a.sup.(1) n/a.sup.(1) .sup.(1)sample could not been measured - particle concentration too low

Example 11: Dynamic Light Scattering (DLS) Analysis of Spray-Freeze-Dried Formulations

[0278] DLS measurements were carried out by using a Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK) instrument. 150 μl of the sample were analyzed in small volume disposable cuvettes (UVette) by using an automated mode for each sample. As a control (TO), the protamine-formulated RNA before spray-freeze drying was used.

[0279] The Malvern Zetasizer Software was used to calculate Z-average diameter, polydispersity index (PDI) and an intensity size distribution (refractive index and viscosity of water was selected in the software). The results are summarized in Table 8 and the respective diagrams are shown in FIG. 10.

TABLE-US-00008 TABLE 8 Dynamic light scattering (DLS) analysis of spray-freeze-dried formulations Z-average Main peak Main peak Derived count Sample diameter [nm] PDI diameter [nm] intensity [%] rate T0 236.4 ± 6.4 0.196 ± 0.003 288.6 ± 6.6 100 ± 0  59,436 ± 32  T-SFD1 252.3 ± 6.3 0.211 ± 0.010 312.3 ± 4.3 100 ± 0  56,480 ± 316  T-SFD2 236.7 ± 7.2 0.203 ± 0.019 294.3 ± 2.9 99.1 ± 1.5 59,888 ± 179  T0-P  4.3 ± 0.7 0.274 ± 0.036  1.3 ± 0.1 76.3 ± 3.0  145 ± 31 T-SFD-P  371.3 ± 16.3 0.278 ± 0.179  315.6 ± 16.6 97.7 ± 3.9 2,646 ± 337

[0280] Z-average and main peak diameter of protamine-formulated RNA that had been spray-freeze-dried (T-SFD1, T-SFD2) were comparable or slightly increased with respect to untreated protamine-formulated RNA (TO).

Example 12: Zeta Potential of Spray-Freeze-Dried Formulations

[0281] Zeta potential measurements were carried out with a Zetasizer Nano Series instrument (Malvern Instruments, Worcestershire, UK). 750 μl of each formulation were analyzed in disposable folded capillary cells. For each sample, 3 zeta potential measurements consisting of 100 sub-runs were performed and the mean value for zeta potential was calculated. The results are shown in Table 9.

TABLE-US-00009 TABLE 9 Zeta potential of spray-freeze-dried formulations Sample Zeta potential [mV] T0 −31.0 T-SFD1 −35.8 T-SFD2 −29.3 T0-P −1.4 T-SFD-P −21.5