Chimeric alkaline phosphatase-like proteins
11746340 · 2023-09-05
Assignee
Inventors
- Willem Raaben (Amersfoort, NL)
- Luigi Johannes Cornelius Jonk (Utrecht, NL)
- Erik Jan Van Den Berg (Vught, NL)
- Andrea Van Elsas (Oss, NL)
- José Luis Millán (San Diego, CA, US)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
A61K38/465
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
International classification
Abstract
The invention relates to improved alkaline phosphatases, pharmaceutical compositions comprising improved alkaline phosphatases and the use of improved alkaline phosphatases for preventing, treating or curing diseases.
Claims
1. A method for improving, reducing or removing symptoms in a subject suffering from a disease accompanied by a local or systemic zinc deficiency comprising administering to the subject: a protein having phosphatase activity, wherein said protein comprises an amino acid sequence of at least 200 consecutive amino acids having at least 90% sequence identity with SEQ ID NO: 5, an amino acid sequence of at least 50 consecutive amino acids having at least 90% sequence identity with SEQ ID NO: 6, and an amino acid sequence of at least 40 consecutive amino acids having at least 90% sequence identity with SEQ ID NO: 7, wherein the full length protein comprises an amino acid sequence having at least 90% sequence identity with the full length amino acid sequence of SEQ ID NO: 1, with the proviso that the amino acid at position 279 is leucine (L), the amino acid at position 328 is valine (V) and the amino acid at position 478 is leucine (L); and wherein said disease is selected from sepsis-associated acute kidney injury, ischemic reperfusion kidney damage or a hypophosphatasia.
2. The method according to claim 1, wherein said hypophosphatasia is selected from the group consisting of perinatal hypophosphatasia, infantile hypophosphatasia, childhood hypophosphatasia, and adult hypophosphatasia.
3. The method according to claim 1, wherein the protein having phosphatase activity comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, with the proviso that the amino acid at position 279 is leucine (L), the amino acid at position 328 is valine (V) and the amino acid at position 478 is leucine (L).
4. The method according to claim 1, wherein the protein having phosphatase activity comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1, with the proviso that the amino acid at position 279 is leucine (L), the amino acid at position 328 is valine (V) and the amino acid at position 478 is leucine (L).
5. The method according to claim 1, wherein the protein having phosphatase activity comprises an amino acid sequence of 300-365 consecutive amino acids having at least 95% sequence identity with SEQ ID NO: 5, an amino acid sequence of 60-65 consecutive amino acids having at least 95% sequence identity with SEQ ID NO: 6, and an amino acid sequence of 50-54 consecutive amino acids having at least 95% sequence identity with SEQ ID NO: 7, wherein the full length protein comprises an amino acid sequence having at least 95% sequence identity with the full length amino acid sequence of SEQ ID NO: 1, with the proviso that the amino acid at position 279 is leucine (L), the amino acid at position 328 is valine (V) and the amino acid at position 478 is leucine (L).
6. The method according to claim 1, wherein the protein having phosphatase activity comprises an amino acid sequence of 350-365 consecutive amino acids having at least 98% sequence identity with SEQ ID NO: 5, an amino acid sequence of 62-65 consecutive amino acids having at least 98% sequence identity with SEQ ID NO: 6, and an amino acid sequence of 52-54 consecutive amino acids having at least 98% sequence identity with SEQ ID NO: 7, wherein the full length protein comprises an amino acid sequence having at least 98% sequence identity with the full length amino acid sequence of SEQ ID NO: 1, with the proviso that the amino acid at position 279 is leucine (L), the amino acid at position 328 is valine (V) and the amino acid at position 478 is leucine (L).
7. The method according to claim 1, wherein the protein having phosphatase activity comprises the amino acid sequence as set forth in SEQ ID NO: 1.
8. The method according to claim 1, wherein the protein having phosphatase activity consists of the amino acid sequence as set forth in SEQ ID NO: 1.
Description
FIGURE LEGENDS
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EXAMPLES
Example 1
(21) Stability of LVL-RecAP in Buffer
(22) Materials:
(23) Human recombinant alkaline phosphatases: 1. sALPI-ALPP-CD (SEQ ID NO: 4) (DOM: 4 Aug. 2008) 2. LVL-recAP (SEQ ID NO: 1) (DOM: 14 Oct. 2011)
Methods:
Zinc Dependency:
(24) The enzyme activity and protein content of both human recombinant alkaline phosphatase batches were determined. Subsequently 100 μg/mL protein solutions were prepared for each condition to determine Zinc dependency for batches sALPI-ALPP-CD and LVL-recAP as outlined in Table 2.
(25) The prepared samples were stored at RT and analysed for enzyme activity at T=0, T=2 h and T=24 h.
(26) TABLE-US-00002 TABLE 2 Conditions of recombinant alkaline phosphatase batches sALPI-ALPP-CD and LVL-recAP to determine their stability (retained activity) in the presence and absence of Zinc and the influence of a chelator (EDTA). Zn BSA Mg Mannitol EDTA Condition (μM) (%) (mM) (%) (mM) 1 0 0 1 1 0 2 0.01 0 1 1 0 3 10 0 1 1 0 4 100 0 1 1 0 5 1000 0 1 1 0 6 0 0 1 1 2 7 0 0 1 1 5 8 0 0 1 1 10 9 0 0 1 1 100 10 50 0.025 1 1 0
(27) Determinations of enzyme activities were according to standard procedures, as indicated in SOP PC001.
(28) Protein concentrations were determined by OD.sub.280 measurements (ε.sub.recAP 1.01 mL/mg/cm OD.sub.280)
(29) Results
(30) TABLE-US-00003 TABLE 3 activity of the different alkaline phosphatases under the conditions as specified in Table 2. sALPI-ALPP-CD LVL-RecAP Diluent t = 0 t = 2 t = 24 t = 0 t = 2 t = 24 1 45.7 53.3 50 60.7 59.8 57.4 2 48.2 53.8 51.6 57.7 57.2 58.1 3 47.3 54.4 51.6 57.7 54.2 52.9 4 46.1 52 53.4 55.3 53.7 55.7 5 48.2 51.2 52.2 56.7 56.3 54.7 6 46.9 24.2 18.7 57.6 54.2* 37.7* 7 47 23.6 20.4 57.6 52.1* 38.9* 8 45 23.2 17.2 56.8 50.3* 35.8* 9 50.9 22.5 16.2 55.6 46.5* 31.2* 10 47.3 55.1 53.6 62.7 57.5 55.4
(31) As clearly shown in Table 3, LVL-RecAP is considerably more stable (i.e. shows more residual enzyme activity; denoted by values with asterisks) in the presence of a metal chelating agent (EDTA) than sALPI-ALPP CD, implicating less dependency of Zn.sup.2+ for its activity.
Example 2
(32) Stability of LVL-RecAP in buffer
(33) Materials:
(34) Human recombinant alkaline phosphatases: 1. sALPI-ALPP-CD (DOM: 4 Aug. 2008) 2. LVL-recAP (DOM: 14-Oct. 2011)
Methods
(35) In a second independent experiment, stability of sALPI-ALPP-CD and LVL-recAP were tested for several conditions as outlined in table 4.
(36) For both recombinant AP batches in this experiment 100 μg/mL protein solutions in 0.025M glycine buffer pH 9.6, human serum and human citrate plasma were prepared for each buffer condition to determine stability.
(37) All the prepared recombinant AP samples were incubated at 37° C. and analysed for enzyme activity at T=0, T=0.5 h, T=1 h, T=2 h and T=24 h.
(38) TABLE-US-00004 TABLE 4 Conditions of recombinant alkaline phosphatase batches sALPI- ALPP-CD and LVL-recAP in glycine buffer to determine their stability (retained activity) in the presence/absence of Zinc and the influence of a Chelator (EDTA or Citrate). Zn BSA Mg Mannitol EDTA Condition (μM) % (mM) (%) (mM) 1 0 0 1 1 0 2 0 0 1 1 2 3 0 0 1 1 10 4 0 0 1 1 100 8 50 0.025 1 1 0
Results
(39) TABLE-US-00005 TABLE 5 activity of the different alkaline phosphatases under the conditions as specified in Table 4. LVL-recAP sALPI-ALPP-CD Condition t = 0 t = 0.5 t = 1 t = 2 t = 24 t = 0 t = 0.5 t = 1 t = 2 t = 240 1 55.1 57.7 54.9 55.2 55.8 63 52.5 53.8 53.8 55.2 2 59.5 52.5 52 51.6* 37.7* 56.5 48 51.6 36.3 23.3 3 50.9 53.5 51.6 49.2* 35.2* 3.3 44.7 43.7 32.9 18.9 4 49.3 43.9 48.7 41.3* 24.2* 49.8 37.9 34.6 29.4 13.3 8 12.2 49.3 53.9 53.1 52.1 55.9 56 57.9 57.5 56
(40) Determinations of enzyme activities were performed according to SOP PC001. Protein concentrations were determined by OD280 measurements (εrecAP 1.01 mL/mg/cm OD280).
(41) As clearly shown in Table 5 and in line with results in Table 3, LVL-RecAP is considerably more stable in the presence of a metal chelating agent (EDTA) than sALPI-ALPP CD (as denoted by values with asterisks), implicating less dependency of Zn.sup.2+ for its activity.
Example 3
(42) Heat Stability and PLP Kinetics of LVL-RecAP
(43) Methods
(44) Protein Expression
(45) Expression plasmids containing secreted, FLAG epitope-tagged sALPI-PLAP-CD and LVL-RecAP were constructed as described previously (Kozlenkov et al. J Biol Chem 277, 22992-22999 (2002) and Kozlenkov et al. J. Bone Miner. Res. 19, 1862-1872 (2004)). The FLAG-tagged enzymes were transiently transfected into COS-1 cells by electroporation, then grown in DMEM medium for 24 h, as previously described (Narisawa et al. Am. J Physiol. Gastrointest. Liver Physiol. 293, G1068-1077 (2007)) when the medium was replaced with serum-free Opti-MEM (Life Technologies). Opti-MEM containing secreted proteins was collected 60 hours after transfection, then filtered through a 2-μm cellulose acetate filter and dialyzed against TBS containing 1 mM MgCl.sub.2 and 20 μM ZnCl.sub.2.
(46) Enzyme Kinetics
(47) To measure the relative catalytic activities of FLAG-tagged enzymes, micro-titer plates were coated with anti-FLAG M2 antibody (Sigma-Aldrich) at 0.2-0.6 μg mL.sup.−1. These plates were incubated with saturating concentrations of FLAG-tagged LVL-RecAP or sALPI-PLAP for 3 h at room temperature, after which plates were washed with PBS, containing 0.008% Tween-80 and the relative activities for PLP were compared for the M2-saturated enzymes.
(48) Hydrolysis of the physiological substrate pyridoxal-5′-phosphate (PLP) (Sigma-Aldrich) was measured at pH 7.4 in standard assay buffer (50 mM Tris-HCl buffer, 100 mM NaCl, 1 mM MgCl.sub.2 and 20 μM ZnCl.sub.2). The concentration of released phosphate was determined using P.sub.i ColorLock Gold (Innova Biosciences) by measuring absorbance at 650 nm (A.sub.650). Standard curves constructed for increasing concentrations of phosphate were linear between 0-50 μM and all experiments were designed to fall within this range of hydrolyzed phosphate concentrations. Molar reaction rates, expressed as [P.sub.i] s.sup.−1 were calculated for the indicated substrate concentration range and were fitted to a one-binding site model (GraphPad Prism) versus [substrate] to calculate K.sub.m (Lineweaver-Burk plots were not applied, because of lack of precision of reciprocal conversions at very low substrate concentrations).
(49) A substrate concentration for PLP of 400 μM was used. The soluble enzyme concentration was about 1 nM and incubation times ranged from 15-30 min, depending on the catalytic efficiency for each enzyme. To ensure steady-state conditions and to correct for non-specific substrate signals in the P.sub.i ColorLock Gold method, an early reading (at 5 min) was subtracted from the later reading, and ΔA.sub.650 was measured on the corresponding P.sub.i standard curve, constructed for each experiment separately. All experiments were carried out three to five times and the derived constants are reported as mean±SD.
(50) To measure the heat stability, FLAG-tagged enzymes were incubated at 65° C. in 1 M DEA (pH 9.8) containing 1 mM MgCl.sub.2 and 20 μM ZnCl.sub.2. Samples were removed at different time points and placed on ice, then residual activity was measured using pNPP using the following method: The activity of bound enzymes was measured as the absorbance at 405 nm (A.sub.405) as a function of time at 25° C., using pNPP (10 mM) as a substrate, at pH 7.4 in 50 mM Tris-HCl buffer, 100 mM NaCl, containing 1 mM MgCl.sub.2 and 20 μM ZnCl.sub.2. Enzymes were also incubated in this buffer for 10 min at increasing temperatures (25-100° C.) and residual activity was measured in the same manner.
(51) Results
(52) Production of FLAG-Tagged Enzymes
(53) To compare the kinetic properties of LVL-RecAP with sALPI-PLAP, a FLAG-tag sequence was added to both cDNAs, as done previously to comparatively study PLAP and TNAP (Kozlenkov et al. J Biol Chem 277, 22992-22999 (2002) and Kozlenkov et al. J. Bone Miner. Res. 19, 1862-1872 (2004)). The cDNAs were expressed in COS-1 cells and culture supernatant containing secreted enzymes was recovered. Successful expression and recovery were confirmed by anti-FLAG antibody western blot analysis.
(54) Kinetics Parameters with Physiological Substrates
(55) The phosphohydrolase properties of LVL-RecAP and sALPI-PLAP were investigated for physiological substrates implicated in inflammation and seizures, specifically the vitamin B.sub.6 vitamer PLP. LVL-RecAP showed lower Km than sALPI-PLAP (30.1 μM vs 60.7 μM) at physiologic pH (7.4), indicating that the improved LVL-RecAP has higher affinity for PLP than sALPI-PLAP at physiologic pH.
(56) Enzyme Stability
(57) The impact of the amino acid mutations in LVL-RecAP on the overall stability of the enzyme was investigated by heat inactivation studies. Although sALPI-PLAP has already highly improved heat resistance (50% inactivation at 77.8° C.), LVL-RecAP was even more resistant to heat inactivation (50% inactivation at 80.6° C.).
Example 4
(58) Pharmacokinetic Distribution of LVL-RecAP in Rats
(59) Part A Radiolabelling with Iodine-125
(60) The sALPI-PLAP-CD protein and the LVL-RecAP protein were radiolabelled with Iodine-125 using the chloramine-T procedure as described by Greenwood et al.* The principle of labelling is based on the “in situ” oxidation of iodide to atomic iodine and its nucleophilic substitution into phenol rings in ortho position of the hydroxyl group of tyrosine residues. *F. C Greenwood, V. M Hunter, H. G Glover, The preparation of 131I labelled growth hormone of high specific radioactivity, Biochem. J. 89 (1963) 114-123
(61) The sALPI-PLAP protein was radioiodinated using the Chloramine-T technique in order to obtain ˜0.5 mCi/mg final specific activity and ˜1 mg/mL (NaCl 0.9%) final concentration.
(62) A.1 Materials sALPI-PLAP Alkaline Phosphatase (496.7 U/mg) and LVL-RecAP (624 U/mg) was provided by AM-Pharma at 6.34 mg/mL concentration in 25% glycerol w/v, 5 mM Tris, 2 mM MgCl.sub.2, 50 μM ZnCl.sub.2, pH 8.0. sALPI-PLAP protein and LVL-RecAP protein were stored at 4° C. Iodine-125 radionuclide was purchased from Perkin Elmer as sodium iodide in 10.sup.−5N sodium hydroxide (specific activity: 643.8 GBq/mg—radionuclide purity: 99.95%). Chloramine-T (N-chloro-p-toluenesulfonamide, MW 227.6 g/mol), trichloroacetic acid, sodium metabisulfite (MW 190.1 g/mol) and tyrosine were purchased from Sigma.
(63) Tris Buffer 5 mM pH 8 was prepared in Chelatec laboratory. NaCl 0.9% was provided by Versol®.
(64) A.2 Method
(65) 850 μg of protein, about 600 μCi of Na.sup.125I, 50 μL of Tris buffer and 10 μL of chloramine-T (404.8 nmol, 50 equivalents/protein) were successively added in a 1.5 mL Lo-Bind eppendorf tube. The reaction was allowed to stir 1 minute at room temperature. 2 μL of the radiolabelling medium was mixed with MBS 5% solution and the radiolabelling efficiency was evaluated by Instant thin layer chromatography (ITLC) with 10% TCA as eluent (alkaline phosphatase at the bottom of the strip and free 125-iodine is eluted at the top of the strip).
(66) After adding 30 μL of tyrosine solution (10 mg/mL in water) to the crude mixture, the iodinated sALPI-PLAP and iodinated LVL-RecAP were purified by gel filtration (G10, GE Healthcare) eluted with NaCl 0.9%. Fractions of 0.2 mL volume were collected in test tubes. Radioactivity in each fraction was measured in an automatic Gamma Counter calibrated for iodine-125 radionuclide (Wallace Wizard 2470—Perkin Elmer). The fractions containing the desired radioiodinated product were pooled. The radiochemical purity of the radiolabelled compound was verified by ITLC.
(67) A.3 Results and Characteristic of Radiolabelled sALPI-PLAP Protein
(68) The efficiency of radiolabelling determined by ITLC was over 85% for both proteins. After G10 purification, radiopurity of the labelling reaction is higher than 97% for both proteins.
(69) The characteristics of radiolabelled sALPI-PLAP solution after G10 purification are summarized in Table 6.
(70) TABLE-US-00006 TABLE 6 Characteristics of .sup.125I-sALPI-PLAP solution after purification .sup.125I-sALPI-PLAP .sup.125I-LVL-RecAP Labelling efficiency 86.36% 85.64% (ITLC) G10 purification Concentration 1.149 1.145 (mg/mL) Specific Activity 0.669 0.544 (mCi/mg) Volumic Activity 0.791 0.637 (mCi/mL) Radiopurity (%) 97.23 97.8
(71) A.4 Alkaline Phosphatase Activity
(72) The enzymatic activity of radiolabelled sALPI-PLAP was assessed via ELISA.
(73) Materials
(74) Alkaline phosphatase colorimetric assay kit (reference ab83369—batch number: GR118166-3). Unlabelled recombinant human alkaline phosphatase diluted at 0.25 mg/mL in NaCl 0.9% Radiolabelled recombinant human alkaline phosphatase diluted at 0.25 mg/mL in NaCl 0.9%
Protocol
(75) The Abcam kit uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow (Lamda max=405 nm) when dephosphorylated by AP. The kit can detect 10-250 μU AP in samples.
(76) The alkaline phosphatase colorimetric assay was performed on unlabelled and radioiodinated sALPI-PLAP. The assay was performed as described in protocol provided by Abcam.
(77) Briefly, a standard curve was generated from 0 to 20 nmol/well of pNPP standard (final volume: 120 μL). 10 μL of AP enzyme solution was added in each well. In parallel, the test sample of sALPI-PLAP was diluted 15000 and 8000 times in assay buffer. 10 and 20 μL of each dilution was added and the final volume was brought to 80 μL with assay buffer. Then, 50 μL of pNPP solution was added to each well containing the test sample.
(78) The standard and sample reactions were incubated 60 min at 25° C. in the dark. All reactions were then stopped with 20 μL of stop solution. The O.D. at 405 nm was measured in a microplate reader.
(79) pNP standard curve was plotted. The sample readings were applied to the standard curve to get the amount of pNP generated by AP sample. AP activity of the test samples can be calculated:
sALPI-PLAP activity (U/mL)=A/V/T×dilution factor of test sample
A: amount of pNP generated by samples (in μmol)
V: volume of sample added in the assay well (in mL)
T: reaction time in minutes
sALPI-PLAP activity (U/mg)=sALPI-PLAP activity (U/mL)/concentration of sALPI-PLAP (mg/mL)
Results
(80) Table 7 summarises the results.
(81) TABLE-US-00007 TABLE 7 Enzymatic activity of unlabelled sALPI-PLAP and .sup.125I-ALPI-PLAP sALPI-PLAP activity (U/mg) Dilution 1/15000 Dilution 1/8000 Mean sALPI-PLAP 474.6 471.6 473.1 .sup.125I-sALPI-PLAP 482.6 475.3 479.0 LVL-RecAP 501.3 530.2 515.8 .sup.125I-LVL-RecAP 539.2 588.5 563.8
(82) The enzymatic activity of unlabelled and radiolabelled sALPI-PLAP is similar to about 475 U/mg. Thus, the activity of sALPI-PLAP is not compromised by the radioiodination using chloramine-T as oxidant.
(83) Part B: Biodistribution Study of .sup.125I-sALPI-PLAP Protein in Healthy Rats
(84) B.1 Materials
(85) The characteristics of the rat strain used in this study are presented below: Species: Sprague Dawley Rats Strain: Crl CD® (SD) IGS BR Source: Charles RIver France Number and sex: 15 males Body weight range/age: Approximately 250 g at the beginning of the study Acclimation period: Five days before treatment Identification method: Cage identification with the group (sacrifice time).
Animal Management Husbandry: The rats were housed in animal facilities before treatment and in radioactivity room after treatment. Food: Freely available rat diet. No fasting period before treatment. Water: Tap water was delivered by polypropylene bottle ad libitum. Housing: Before treatment, animals were housed in groups of three in polycarbonate cages in standard conditions, identified by a card indicating the study number, the animal number, the sex, and the dates of beginning and end of the study. Animals designated for excretion balance were transferred in metabolism cages (one by cage) after treatment. Environmental: The temperature was recorded every day. The temperature of the room was between 22 and 24° C. The artificial light cycle was controlled using an automatic timer (10 hours of light, 14 hours of dark). Personnel: Associates involved were appropriately qualified and trained. Selection: Animals were examined at receipt by the study director. Only healthy animals were selected. Particular attention was paid to any sign of inflammatory reaction in the animals (e.g. abscess; skin inflammation etc).
(86) B.2 Dosing Solution of .sup.125I-sALPI-PLAP and 125I-LVL-RecAP
(87) The Iodinated sALPI-PLAP and LVL-RecAP solutions were diluted at 0.25 mg/mL in NaCl 0.9% just prior to in vivo administration.
(88) B. 3 Study Design
(89) The study design is presented in Tables 8 and 9.
(90) TABLE-US-00008 TABLE 8 Study design for .sup.125I-sALPI-PLAP in vivo distribution Number of Blood sampling Sacrifice Group animals time time Biodistribution A1 3 males 2 mn, 10 mn 30 mn Blood and organs A2 3 males 5 mn, 45 mn 2 h Blood and organs A3 3 males 15 mn, 4 h 6 h Blood and organs A4 3 males 1 h, 3 h, 18 h 24 h Blood and organs A5* 3 males NA 48 h Blood and organs *housed individually in metabolism cages with urine and faeces collection at 24 h and 48 h
(91) TABLE-US-00009 TABLE 9 Study design for .sup.125I-LVL-RecAP in vivo distribution Number of Blood sampling Sacrifice Group animals time time Biodistribution B1 3 males 2 mn, 10 mn 30 mn Blood and organs B2 3 males 5 mn, 45 mn 2 h Blood and organs B3 3 males 15 mn, 4 h 6 h Blood and organs B4 3 males 1 h, 3 h, 18 h 24 h Blood and organs B5* 3 males NA 48 h Blood and organs
(92) B.4 Administration
(93) At the time of the experiment, the mean weight of the Sprague Dawley rats was about 240 g. Unanesthetized rats were injected intravenously in lateral tail vein (left) at a dose level of 400 μg/kg corresponding to a quantity of 96 μg protein per rat and an activity of about 58 μCi per rat. The volume of injection was about 380 μL. The individual dose volumes were calculated using individual body weight of each rat on the day of treatment. Rats were placed in a contention device. In order to dilate the tail blood vessels, the tail was dipped in warm water (45° C.) and then disinfected with alcohol. The dosing solution was slowly injected in the tail vein. In order to calculate the actual dose received by each rat, the syringes were weighed before and after treatment and an aliquot of dosing solution was counted in a Gamma Counter.
(94) B.5 Distribution in Various Organs
(95) At the time of sacrifice, the animals were anaesthetized by intraperitoneal injection of 2.5 mL/kg body weight of a mixture ketamine hydrochloride (50 mg/mL) and xylazine hydrochloride (20 mg/mL) in PBS. Rats were then rapidly sacrificed by exsanguination via intracardiac puncture. The organs of interest were harvested followed by the cutting in pieces for organs weighting more that 2 g such as liver, stomach, small intestine and colon. Each piece was thereafter rinsed with physiological serum prior to be wiped with soft paper tissue, weighed and counted separately. The selected organs were liver, kidneys, heart, lungs, spleen, skeletal muscle, femur, brain, thyroid, stomach, small intestine with content, colon with content, skin and perirenal fat.
(96) The counting of tissue radioactivity was performed in an automatic gamma counter (Wallace Wizard 2470—Perkin Elmer) calibrated for Iodine-125 radionuclide (efficiency: 74%—counting time: 10 sec).
(97) The concentration of radioactivity in the organs/tissues is expressed as percentage of the injected dose per gram of tissue (% ID/g). Data analysis includes the percentage of the injected dose (% ID) and equivalent quantity of protein per organ or tissue. For well-defined organs, these were calculated using the radioactivity counted in whole organ. For blood, this was approached assuming that blood accounts for 6.4% of total body weight.
(98) In addition, the ratio between the radioactivity retained in the tissues and the radioactivity in blood was calculated (ratio organ/blood). Finally, the ratio between the organ/blood ratio of sALPI-PLAP and the organ/blood ratio of LVL-RecAP was calculated (
(99) B.6 Distribution in Rat Blood and Serum
(100) Blood and Serum Radioactive Level
(101) At the time of sacrifice, the blood samples were obtained from exsanguinations via intracardiac puncture on anaesthetized rat by intraperitoneal injection of a mixture of ketamine hydrochloride and xylazine hydrochloride in PBS.
(102) At the other time points indicated in the study design (table 8 and 9), the blood was withdrawn from the lateral tail vein (right) using a 23-gauge butterfly needle without anesthesia.
(103) Each blood sample was collected into preweighed Microvette tubes with clotting activator (Sarstedt). The tubes were weighed and the radioactivity was measured in the Gamma counter.
(104) Blood samples were incubated at room temperature for 30 min and then, they are centrifuged for 5 minutes at 10 000 g to prepare serum. Serum was collected into pre-weighed tubes and counted in the Gamma counter.
(105) The concentration of radioactivity in blood and serum is expressed as percentage of the injected dose and equivalent quantity of sALPI-PLAP per mL.
(106) Data analysis includes the percentage of the injected calculated for the total blood and serum.
(107) Tables 10 and 11 show the ratio of radioactivity measured in different organs in relation to blood serum radioactivity of sALPI-PLAP and LVL-RecAP respectively.
(108) TABLE-US-00010 TABLE 10 Organ/Blood ratio for sALPI-PLAP ORGANS 0.5 H 2 H 6 H 24 H 48 H Thyroid/ 0.1624 ± 0.0157 4.6771 ± 0.4433 31.745 ± 5.175 208.16 ± 23.74 733.85 ± 79.16 Trachea Skin 0.0307 ± 0.0035 0.2842 ± 0.0134 0.4661 ± 0.1617 0.9448 ± 0.2565 1.0787 ± 0.3686 Kidneys 0.2076 ± 0.0011 0.3438 ± 0.0310 0.3535 ± 0.0219 0.3250 ± 0.0467 0.4299 ± 0.0219 Stomach 0.0446 ± 0.0016 1.0699 ± 0.1665 3.0622 ± 0.6977 1.4478 ± 0.1200 3.5819 ± 0.9018 Spleen 0.5388 ± 0.0796 0.4133 ± 0.0322 0.2880 ± 0.0283 0.2256 ± 0.0270 0.2273 ± 0.0254 Liver 5.2403 ± 0.1943 1.9917 ± 0.0347 0.4811 ± 0.0877 0.3219 ± 0.0325 0.4676 ± 0.0295 Heart 0.1824 ± 0.0136 0.2259 ± 0.0198 0.2605 ± 0.0217 0.2791 ± 0.0387 0.2809 ± 0.0165 Lungs 0.1777 ± 0.0119 0.2767 ± 0.0318 0.3397 ± 0.0730 0.3833 ± 0.0091 0.3937 ± 0.0251 Skeletal 0.0334 ± 0.0033 0.0978 ± 0.0193 0.0842 ± 0.0178 0.0876 ± 0.0038 0.0947 ± 0.0150 muscle Fat 0.0095 ± 0.0023 0.0261 ± 0.0070 0.0509 ± 0.0111 0.0615 ± 0.0065 0.0874 ± 0.0153 Brain 0.0160 ± 0.0031 0.0257 ± 0.0011 0.0213 ± 0.0011 0.0253 ± 0.0025 0.0408 ± 0.0063 Femur 0.1886 ± 0.0217 0.1991 ± 0.0131 0.1901 ± 0.0155 0.1602 ± 0.0119 0.1612 ± 0.0132 Small 0.1882 ± 0.0247 0.8440 ± 0.1896 1.1055 ± 0.0827 1.0729 ± 0.1268 0.6329 ± 0.1044 intestine Colon 0.0227 ± 0.0028 0.0777 ± 0.0077 0.4150 ± 0.0489 0.3473 ± 0.0596 0.3991 ± 0.0964
(109) TABLE-US-00011 TABLE 11 Organ/Blood ratio for LVL-RecAP ORGANS 0.5 H 2 H 6 H 24 H 48 H Thyroid/ 0.5934 ± 0.0892 7.3638 ± 1.5049 52.934 ± 13.475 462.83 ± 34.395 1075.2 ± 118.65 Trachea Skin 0.0942 ± 0.0012 0.4110 ± 0.0485 0.6843 ± 0.0810 1.2130 ± 0.1243 1.9213 ± 0.8965 Kidneys 0.5793 ± 0.0187 0.6425 ± 0.0555 0.6428 ± 0.0098 0.8238 ± 0.0826 1.2481 ± 0.1089 Stomach 0.2262 ± 0.1021 1.5983 ± 0.3099 6.2917 ± 3.5698 3.0042 ± 1.2012 0.9930 ± 0.3279 Spleen 2.2771 ± 0.1172 1.0977 ± 0.1190 0.5831 ± 0.0292 0.3673 ± 0.0796 0.3623 ± 0.0429 Liver 7.6513 ± 0.9295 3.8409 ± 0.0277 0.6131 ± 0.0900 0.5869 ± 0.0967 0.7940 ± 0.0133 Heart 0.2030 ± 0.0105 0.2762 ± 0.0096 0.2780 ± 0.0316 0.2630 ± 0.0138 0.3144 ± 0.0023 Lungs 0.3007 ± 0.0201 0.3740 ± 0.0446 0.4332 ± 0.0111 0.3966 ± 0.0235 0.4515 ± 0.0220 Skeletal 0.0462 ± 0.0072 0.0997 ± 0.0142 0.1036 ± 0.0251 0.0784 ± 0.0113 0.0953 ± 0.0080 muscle Fat 0.0298 ± 0.0078 0.0380 ± 0.0063 0.0495 ± 0.0034 0.0781 ± 0.0155 0.0970 ± 0.0245 Brain 0.0261 ± 0.0052 0.0389 ± 0.0085 0.0253 ± 0.0028 0.0288 ± 0.0016 0.0646 ± 0.0100 Femur 0.3764 ± 0.0102 0.2973 ± 0.0098 0.2460 ± 0.0149 0.1804 ± 0.0325 0.1746 ± 0.0370 Small 0.1573 ± 0.0049 0.5625 ± 0.0393 1.0308 ± 0.1134 0.9402 ± 0.0935 0.6612 ± 0.1003 intestine Colon 0.0361 ± 0.0047 0.1049 ± 0.0271 0.3658 ± 0.0693 0.5353 ± 0.1967 0.4787 ± 0.0712
Example 5
(110) Akp2−/− Mouse Model of Infantile Hypophosphatasia
(111) The Akp2−/− mice model of infantile hypophosphatasia is known in the art (J Dent Res 90(4):470-476, 2011). In short, Akp2−/− mice were created by insertion of the Neo cassette into exon 6 of the mouse TNALP gene (Akp2) via homologous recombination to functionally inactivate the Akp2 gene, resulting in no detectable TNALP mRNA or protein.
(112) Animal use and tissue collection procedures followed approved protocols from the Sanford-Burnham Medical Research Institute Animal Ethics Committee. Animals were treated with either vehicle (N=10), 1 mg LVL-RecAP/kg/day (N=10); 8 mg LVL-RecAP/kg/day (N=8) or 16 mg LVL-RecAP/kg/day (N=10). Survival was measured and skeletal development assessed. Mineralization of kidneys was assessed. Plasma PPi, plasma pyridoxal, plasma calcium and phosphate levels were measured, femur and/or tibia length for the different treatment groups were measured. MicroCT data was colleted for analysis of residual hyperosteodosis at each dose.
(113) Results
(114) Enhanced long-term survival for animals treated at 16 mg/kg/day (
Example 6
(115) Ischemia Reperfusion in a Pig Kidney Model
(116) Materials and Methods
(117) Vehicle, Control and Test Article Information
(118) Control and Test Article Preparation
(119) Fresh control article, LVL-RecAP Diluent Solution (placebo), was prepared for use on study prior to each dose administration and was stored refrigerated at 2 to 8° C. when not in use.
(120) The test article, LVL-RecAP, was used as received. No adjustment was made for purity when preparing the test article formulations. Formulations of the test article were prepared by mixing with the appropriate volume of sterile saline to achieve nominal concentrations of 0, 0.96, or 4.8 mg/mL. Formulations were prepared prior to each dose administration under a laminar flow hood using sterile equipment and aseptic techniques. The formulations were dispensed into the appropriate number of amber glass serum bottles, kept on ice prior to use and were used for dosing within 2 hours of preparation. On occasion, additional preparations were made as necessary during the course of the study.
(121) Analysis of Dosing Formulations
(122) Duplicate 0.5 mL samples of the final dosing formulation were collected on prior to dosing each day of preparation on Day 0 (Groups 3 to 7) and Day 7 (Group 7). Samples were collected from the middle strata and were stored frozen (−50 to −90° C.) for possible future analysis.
(123) Test System Information
(124) Animal Acquisition and Acclimation
(125) Male experimentally naïve Domestic Yorkshire crossbred swine (farm pigs) (approximately 8 to 10 weeks of age, at receipt) were received from Midwest Research Swine, Gibbon, Minn. During the 10 to 28 day acclimation period, the animals were observed daily with respect to general health and any signs of disease. Ova and parasite evaluations on stool samples were performed, and all results were negative for animals placed on study.
(126) Randomization, Assignment to Study, and Maintenance
(127) Using separate simple randomization procedures animals (weighing 12.5 to 25.0 kg at randomization) were assigned to the control and treatment groups identified in the following Table 12.
(128) TABLE-US-00012 TABLE 12 Group Assignments Group Number of Male Animals.sup.a Number Dose Level Initial Evaluated 1 Control 7 6 2 Sham.sup.b 7 6 5 0.32 mg/kg 7 6 6 1.6 mg/kg 7 7 7 0.32 mg/kg/day.sup.c 7 7 .sup.aOn Day 0, animals underwent a surgical procedure during which the left or right kidney was removed and the contralateral renal artery was occluded for 45 minutes. Following occlusion, the vessel was allowed to reperfuse. Seven animals were submitted for surgery in each group with the intent to achieve six animals on study. .sup.bSham animals underwent the surgical procedure during which the left kidney was removed. However, renal occlusion and reperfusion were not conducted. .sup.cOn Day 0, half of the dose was administered prior to reperfusion and the remaining half of the dose was administered at 8 ± 2 hours post-reperfusion. A full dose was administered daily for the remainder of the in-life period.
(129) Animals selected for study were as uniform in age and weight as possible. A veterinarian assessed the health of the animals prior to placement on study. Extra animals obtained for the study, but not placed on study, were transferred to the stock colony.
(130) Each animal was assigned an animal number used in the Provantis™ data collection system and was implanted with a microchip bearing a unique identification number. Each animal was also identified by a vendor ear tag. The individual animal number, implant number, ear tag number, and study number comprised a unique identification for each animal. Each cage was identified by the animal number, study number, group number, and sex.
(131) The animals were individually housed in runs with raised flooring or stainless steel mobile cages with plastic coated flooring. This type of housing provided adequate room for exercise for these animals. Animal enrichment was provided according to MPI Research SOP. Fluorescent lighting was provided for approximately 12 hours per day. The dark cycle was interrupted intermittently due to study related activities. Temperature and humidity were continuously monitored, recorded, and maintained to the maximum extent possible within the protocol designated ranges of 61 to 81° F. and 30 to 70%, respectively. The actual temperature and humidity findings are not reported, but are maintained in the study file.
(132) Diet (Certified Lab Diet® #5K99, PMI Nutrition International, Inc.) was offered via limited feedings, except during designated periods. Food enrichment, including fiber bits or tablets, was offered as needed.
(133) Surgical Procedures
(134) Procedure-related Medications
(135) The following Table 13 presents the procedure-related medications and dose levels used during the course of the study.
(136) TABLE-US-00013 TABLE 13 Procedure-related Medications and Dose Levels Interval, Dose Level, and Route Surgery Daily Medication (Day 0) Postsurgery Acepromazine maleate 0.1 mg/kg IM — Atropine sulfate 0.05 mg/kg IM — Telazol 5 to 8 mg/kg IM — Isoflurane To effect by — inhalation Buprenorphine 0.02 mg/kg IM TID 0.02 mg/kg IM TID x 3 days Ketoprofen 3 mg/kg IM 3 mg/kg IM SID x 3 days Cefazolin 25 mg/kg IV — Ceftiofur 2.2 mg/kg IM 2.2 mg/kg IM SID x 3 days Lactated Ringer's 10 to 15 mL/kg/hr — solution (LRS) IV Marcaine 2 mg/kg INF — 0.9% NaCl As needed for — irrigation IV—Intravenous IM—Intramuscular INF—Infused into incision SID—Once daily TID—Twice Daily (every 6 to 9 hours)
(137) Pre- and post-operative procedures were conducted in accordance with MPI Research SOP. The animals were fasted for at least 8 hours prior to surgery and anesthesia was induced and maintained as indicated in Table 13. Body temperature was maintained at 37±3° C. Prior to surgery, ultrasounds were performed to determine if renal cysts were present. If no cysts were observed, the left kidney was removed and the right kidney was treated as described below. If cysts were present on one kidney, that kidney was removed and the contralateral kidney underwent the occlusion procedure. If cysts were present in both kidneys, the animal was removed from study without undergoing the surgical procedure.
(138) Surgical Procedure
(139) Renal ischemia/reperfusion injury was induced using the procedure published by Lee et al (J. Vet. Med. Sci 72(1): 127-130). Once anesthetized, all animals were placed in dorsal recumbency and the surgical sites were prepared with alternating wipes of chlorhexidine scrub and solution. A midline laparotomy was performed to expose both kidneys. Based on the ultrasound findings, the left or right kidney was removed.
(140) For Group 2 animals (Sham), the inserted lap sponges were then removed and accounted for and the abdomen was lavaged with warm sodium chloride. The abdomen was closed in a routine manner and the skin was closed with skin staples and tissue glue. The animals were then allowed to recover.
(141) For all other animals, after removal of the designated kidney, the remaining renal vessels were isolated and retracted using vessel loops. The vessel loops were used to occlude the vessels for 45 (±1) minutes, after which the vessels were allowed to reperfuse. An intravenous bolus dose of the control or test article was administered at a dose volume of 0.333 mL/kg (a half dose of 0.167 mL/kg was administered to Group 7 animals) just prior to reperfusion. For all Group 1, 5 and 6 animals (control, 0.32, and 1.6 mg/kg), the implanted lap sponges were then removed and accounted for, the abdomen was lavaged and closed as described above, and the animals were allowed to recover. For all Group 7 animals (0.32 mg/kg/day), an incision was made in the groin and the left or right femoral vein was isolated. A catheter was advanced into the vessel and the catheter was tunneled under the skin and exteriorized through an incision made on the thorax. A port was attached and anchored to the muscle with non-absorbable suture. The implanted lap sponges were then removed and accounted for, the abdomen was lavaged and closed as described above, and the animals were allowed to recover.
(142) Test or Control Article Administration
(143) On Day 0, the control or test article was intravenously administered to all Group 1, 5, and 6 animals just prior to reperfusion at a full dose of 0, 0.32, and 1.6 mg/kg, respectively. All doses were administered at a dose volume of 0.333 mL/kg. Also on Day 0, the test article was intravenously administered to all Group 7 animals at a combined dose level of 0.32 mg/kg in two separate half doses of 0.16 mg/kg at a dose volume of 0.167 mL/kg. The first dose was administered just prior to reperfusion and the second dose was administered at approximately 8 (±2) hours post-reperfusion. Full doses of 0.32 mg/kg/day (0.333 mL/kg) were administered to all Group 7 animals on Days 1 to 7 at approximately the same time of the day as the initial Day 0 dose (±2 hours).
(144) Statistics
(145) Table 14 below defines the set of comparisons used in the statistical analyses described in this section.
(146) TABLE-US-00014 TABLE 14 Table of Statistical Comparisons Reference Comparison Group Groups 1 2, 5, 6, 7 2 5, 6, 7 5 7
(147) The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint and group. For serum creatinine concentrations, treatment groups were compared to the reference groups using Repeated Measures Analysis of Covariance (RMANCOVA).
(148) Repeated Measures Analysis of Covariance (RMANCOVA)
(149) For endpoints measured at three or more post-test time intervals, a repeated measures analysis (mixed model) was conducted. For each endpoint, the model tested for the effects of treatment, time, and the interaction of treatment and time. Pre-test data (last measurement before dosing) were included in the model as a covariate.
(150) If there was no significant (p>0.05) treatment by time interaction, the treatment main effect was evaluated. If the treatment effect was not significant (p>0.05), the results were deemed not significant and no further analyses was conducted on the variable. If the treatment effect was significant (p<0.05), linear contrasts were constructed for pairwise comparison of each treatment group with the reference group. If the interaction was significant (p<0.05), each treatment group was compared to the appropriate reference group through the simple effect of ‘treatment’ for each time point. These simple effect pairwise comparisons were obtained from the ‘treatment by time’ interaction.
(151) Results of all pair-wise comparisons are reported at the 0.05 and 0.01 significance levels. All tests were two-tailed tests.
(152) Results
(153) Serum Creatinine
(154) As shown in
(155) As illustrated in
Example 7
(156) Safety Testing in Human
(157) Material and Methods
(158) Objectives
(159) To evaluate the safety and tolerability of single and multiple doses of recombinant improved alkaline phosphatase (LVL-recAP) administered by intravenous (i.v.) infusion in healthy subjects.
(160) To determine the pharmacokinetics (PK) of LVL-recAP after i.v. infusion of single and multiple doses of LVL-recAP in healthy subjects.
(161) Design and Treatments
(162) This is a 2-part, single-center study in a planned number of 50 healthy subjects. Part A will be a randomized, double-blinded, placebo-controlled, single ascending dose (SAD) study in up to 4 sequential groups of 8 healthy male and female subjects each (6 on LVL-recAP and 2 on placebo). An attempt will be made to include in each treatment group an equal number of male and female subjects, with a minimal of 2 and a maximum of 4 females per group. A single dose of LVL-recAP or placebo will be administered by a 1-hour i.v. infusion. Part B will be a randomized, double-blinded, placebo controlled, multiple ascending dose (MAD) study in up to 2 groups of 9 healthy male and female subjects each (6 on LVL-recAP and 3 on placebo). An attempt will be made to include in each treatment group an equal number of male and female subjects, with a minimal of 2 and a maximum of 4 females per group. Subjects will receive a 1-hour i.v. infusion of LVL-recAP or placebo on Days 1, 3 and 5. The following treatments will be administered:
(163) Part A
(164) Group 1: 1-hour infusion of 200 U/kg LVL-recAP
(165) Group 2: 1-hour infusion of 500 U/kg LVL-recAP
(166) Group 3: 1-hour infusion of 1000 U/kg LVL-recAP
(167) Group 4: 1-hour infusion of 2000 U/kg LVL-recAP
(168) Part B
(169) Group 5: 1-hour infusions of 500 U/kg LVL-recAP on Days 1, 2 and 3
(170) Group 6: 1-hour infusions of 1000 U/kg LVL-recAP on Days 1, 2 and 3
(171) After completion of Day 9 of Group 1 and Day 4 of Group 2 of Part A, an interim PK evaluation will be performed.
(172) Depending of the results, infusion and PK sampling schedules may be adjusted for the remaining SAD and MAD groups.
(173) In this first-in-human study, the subjects participating in the lowest dose level in Part A (Group 1) will be dosed according to a sentinel dosing design to ensure minimal risk. This means that initially 2 subjects will be dosed. One of these subjects will receive the active medication LVL-recAP and the other subject will receive placebo. If the safety and tolerability results of the first 24 hours following dosing for the initial subjects are acceptable to the Principal Investigator, the other 6 subjects of the lowest dose level will be dosed in a placebo controlled randomized manner (5 active and 1 placebo).
(174) Observation Period
(175) Part A: from Day −1 until 48 hours (Day 3) after drug administration. Short ambulant visits to the clinical research centre on Days 4, 6, 9 and 15
(176) Part B: from Day −1 until 48 hours (Day 7) after last drug administration. Short ambulant visits to the clinical research centre on Days 8, 10, 13 and 19 Subjects will be screened for eligibility within 3 weeks prior to the (first) drug administration of each group of the study.
(177) Follow-up examinations will take place on Day 15 (Part A) and Day 19 (Part B).
(178) Subjects
(179) Part A: 32 healthy male and female subjects
(180) Part B: 18 healthy male and female subjects
(181) Main Criteria for Inclusion
(182) Gender: male or female
(183) Age: 18-55 years, inclusive
(184) Body Mass Index (BMI): 18.0-30.0 kg/m2, inclusive
(185) Study Drug
(186) Active Drug
(187) Active substance: LVL-recAP, an improved recombinant form of endogenous human alkaline phosphatase (AP)
(188) Activity: a hydrolase enzyme responsible for dephosphorylation of mono-esters of phosphoric acid
(189) Indication: Acute kidney injury
(190) Strength: 600, 1500, 3000 and 6000 U/mL
(191) Dosage form: i.v. infusion
(192) Manufacturer: pharmacy of PRA
(193) Placebo
(194) Substance: 20 mM citrate, 250 mM sorbitol, 2 mM MgCl2, 50 μM ZnCl2, pH 7.0
(195) Activity: none
(196) Indication: not applicable
(197) Strength: not applicable
(198) Dosage form: i.v. infusion
(199) Manufacturer: Nova Laboratories Ltd, Gloucester Crescent, Wigston, Leicester, LE18 4YL, UK
(200) Criteria for Evaluation
(201) Safety: adverse events (AEs), vital signs (including supine systolic and diastolic blood pressure, pulse, body temperature, respiratory rate), 12-lead electrocardiogram (ECG), continuous cardiac monitoring (telemetry), clinical laboratory (including clinical chemistry [AP is considered a PK parameter], hematology and urinalysis) tests, physical examination and anti-drug antibodies (ADA)
Pharmacokinetics PK parameters based on analysis of serum concentrations of LVL-recAP and AP activity.
Results
(202) The dosing and observation time periods for all dosing groups have been concluded and no serious adverse events were observed in any of the groups. The analyses of all parameters measured is ongoing.
Example 8
(203) Materials and Methods
(204) Mice
(205) The generation and characterization of the Alp1.sup.−/− mice has been reported previously (Narisawa et al., 1997). Alp1.sup.−/− mice phenocopy infantile HPP, including global deficiency of TNAP, PP.sub.i accumulation and mineralization defects (Fedde et al., 1999; Narisawa et al., 2001; Anderson et al., 2004; Millan et al., 2008). Dietary supplementation with vitamin B6 briefly suppresses seizures and extends lifespan until postnatal days 18-22 but hypomineralization and accumulation of osteoid continue to worsen with age (Narisawa et al., 1997; Fedde et al., 1999; Narisawa et al., 2001; Millán et al., 2008). Therefore, all animals (breeders, nursing mothers, pups, and weanlings) in this study were given free access to modified laboratory rodent diet 5001 containing increased levels (325 ppm) of pyridoxine. Genotyping was performed by PCR on genomic DNA as previously described (Yadav et al., 2011). The Institutional Animal Care and Use Committee (IACUC) approved all animal studies.
(206) Soluble Chimeric Human Alkaline Phosphatase (LVL-RecAP)
(207) A solution of LVL-RecAP at 10.1 mg/ml in 25% glycerol w/v, 5 mM Tris/HCl, 2 mM MgCl.sub.2, 50 μM ZnCl.sub.2, and at pH 8.0 was used. The enzyme had a purity of >99.99% as determined by high-pressure liquid chromatography.
(208) Dose Response Study with LVL-RecAP
(209) Alp1.sup.−/− mice were divided into 5 cohorts: Vehicle-treated: Alp1.sup.−/− mice treated with vehicle (n=14) only; LVL-RecAP1: Alp1.sup.−/− mice treated with LVL-RecAP at 1 mg/kg/day (n=14); LVL-RecAP8: Alp1.sup.−/− mice treated with LVL-RecAP at 8 mg/kg/day (n=12); and LVL-RecAP16: Alp1.sup.−/− mice treated with LVL-RecAP at 16 mg/kg/day (n=10). Wild-type littermates of Alp1.sup.−/− mice served as reference animals and did not receive injections (n=14). The vehicle or LVL-RecAP cohorts were injected daily SC into the scapular region. Injections were administered between 8:00 and 11:00 AM. Volumes administered were calculated based on body weight measured prior to injection. All treatments began on postnatal day 1, and were repeated daily for up to 53 days or until the time of necropsy.
(210) Sample Collection
(211) Necropsy was performed on postnatal day 53 (p53), 24 h after the final injection of LVL-RecAP for those animals that completed the experimental protocol, or sooner for those animals that appeared terminally ill. Avertin was administered intraperitoneally prior to euthanasia. Blood was collected into lithium heparin tubes by cardiac puncture. Necropsy consisted of a gross pathology examination and x-rays.
(212) Radiography and Microcomputed Tomography (μCT)
(213) Radiographic images of skeletons were obtained with a Faxitron MX-20 DC4 (Chicago, Ill., USA), using an energy of 20 kV. Hemi-mandibles were scanned at 30 kV. Whole dissected skulls from P21 mice were fixed, then scanned at an 18 μm isotropic voxel resolution using the eXplore Locus SP μCT imaging system (GE Healthcare Pre-Clinical Imaging, London, ON, Canada). Measurements were taken at an operating voltage of 80 kV and 80 mA of current, with an exposure time of 1600 ms using the Parker method scan technique, which rotates the sample 180 degrees plus a fan angle of 20 degrees. Scans were calibrated to a hydroxyapatite phantom and 3D images were reconstructed at an effective voxel size of 18 μm.sup.3. A fixed threshold of 1400 Hounsfield Units was used to discriminate mineralized tissue. Regions of interest (ROI's) for parietal and frontal bones were established as 1 mm in length, 1 mm in width, depth equivalent to thickness of bone and position starting at a 0.75 mm distance from sagittal and coronal sutures, as previously described (Liu et al., 2014). Parameters of bone volume, density and structure were measured using Microview version 2.2 software (GE Healthcare Pre-Clinical Imaging, London, ON) and established algorithms (Meganck et al., 2009; Umoh et al., 2009). Student's t-tests comparing quantitative results were performed to establish statistically significant differences between genotypes. μCT bone data were analyzed and are reported in accordance with the recommendations of Bouxsein et al. 2010 (Bouxsein et al., 2010).
(214) For dental imaging, dissected hemi-mandibles were scanned on a Scanco Medical μCT 50 (Scanco Medical AG, Brüttisellen, Switzerland) at 10 μm voxel size. Mandible z-stacks were exported as DICOM files and reoriented using ImageJ software (1.48r), with comparable coronal, sagittal, and transverse planes of section chosen for comparison. For quantitative analysis, mandibles were scanned on a Scanco Medical μCT 35 at 6 μm voxel size, 55 KVp, 145 mA, with 0.36 degrees rotation step (180 degrees angular range) and a 400 ms exposure per view. Scanco μCT software (HP, DECwindows Motif 1.6) was used for 3D reconstruction and image viewing. After 3D reconstruction, crown, enamel, roots, and alveolar bone volumes were segmented using global threshold 0.6 g/cc. Total volume (TV), bone (mineralized tissue) volume (BV), and tissue mineral density (TMD) were measured for the whole crown and separately for enamel, root dentin, and alveolar bone in the furcation region. For enamel and root, thickness was also measured.
(215) Histological Analyses
(216) Bone samples were cleaned, fixed in 4% paraformaldehyde/phosphate buffered saline for 3 days at 4° C., and then transferred to 70% ethanol for storage at 4° C. Plastic sections were prepared as described previously (Yadav et al., 2012). Von Kossa and Van Gieson trichrome staining was performed on plastic sections as described previously (Narisawa et al., 1997). Von Kossa or Van Gieson-stained sections were scanned by ScanScopeXT system (Aperio, Vista, Calif., USA), and images were analyzed by using the Bioquant Osteo software (Bioquant Osteoanalysis Co., Nashville, Tenn., USA). Left hemi-mandibles used for histology were fixed in Bouin's solution for 24 h, and then demineralized in AFS solution (acetic acid, formaldehyde, sodium chloride), and embedded in paraffin for serial sectioning, as described previously (Foster, 2012). For picrosirius red staining, deparaffinized tissue sections were stained with 0.2% aqueous solution of phosphomolybdic acid hydrate, 0.4% Direct red 80, and 1.3% 2,4,6-trinitrophenol (Polysciences, Inc., Warrington, Pa.), as described previously (Foster, 2012). Picrosirius red-stained sections were observed under polarized light for photomicrography.
(217) Biomechanical Testing
(218) After removal of muscle tissue, lengths of the femur, tibia, humerus and radius were measured with a caliper. Bones were frozen, wrapped in gauze containing a saline solution to avoid dehydration. Isolated femurs and tibias were assessed with three-point bending test by using the Instron 1101 universal material testing machine as described previously (Huesa et al., 2011). Bones were slowly thawed and held at room temperature prior to testing. The intact femurs and tibias were placed in the testing machine on two supports separated by a distance of 15 mm and load was applied to the middle of the diaphysis, thus creating a three-point bending test at a speed of 2 mm min.sup.−1.
(219) PP.sub.i Assay
(220) PP.sub.i concentrations in plasma were determined by differential adsorption on activated charcoal of UDP-D[6-3H]glucose (Amersham Pharmacia) from the reaction product 6-phospho [6-3H]gluconate, as described (Hessle et al., 2002; Yadav et al., 2014).
(221) Statistics
(222) Considering that the different concentrations of LVL-RecAP resulted in different survival rates, it was not possible to compare the three treatment cohorts in an age-matched fashion. For this reason, Student t unpaired, parametric, two-tailed test was performed to compare the treated Alp1.sup.−/− mice to the WT cohort. Differences were considered significant when p<0.05. In order to compare the differences in the survival curves among the treatment cohorts, the Gehan-Breslow-Wilcoxon test was performed.
(223) Results
(224) Increased Survival and Body Weight in LVL-RecAP-Treated Alp1.sup.−/− Mice
(225) Survival in mice receiving 8 mg/kg/day (LVL-RecAP8) or 16 mg/kg/day (LVL-RecAP16) of LVL-RecAP was significantly improved compared to the vehicle-treated and the 1 mg/kg/day (LVL-RecAP1) cohort (p=0.001) (
(226) Alp1.sup.−/− animals weigh less than their WT littermates, starting around p7. Treatment with 1 mg/kg/day LVL-RecAP led to a statistically significant increase in body weight compared to vehicle-treated mice, and a non-significant difference compared to WT littermates at 18 days of treatment (
(227) LVL-RecAP Treatment Improves the Skeletal Phenotype of Alp1.sup.−/− Mice
(228) Radiographs of untreated Alp1.sup.−/− mice (
(229) To assess the degree of improvement of osteomalacia, we performed a histomorphometric analysis of plastic-embedded undecalcified sections of hindlimbs of LVL-RecAP-treated and WT control mice (
(230) Absence of Craniofacial Abnormalities in LVL-RecAP-Treated Alp1.sup.−/− Mice
(231) Alp1.sup.−/− mice feature craniofacial shape abnormalities and coronal suture fusion (Liu et al., 2014). To determine the extent to which the craniofacial skeleton is affected by treatment in Alp1.sup.−/− mice, we performed μCT-based analyses of frontal and parietal cranial bones. Results at p21 show that both frontal and parietal bones of vehicle treated Alp1.sup.−/− mice were significantly reduced in bone volume fraction, bone mineral content, bone mineral density, tissue mineral content and tissue mineral density when compared to WT or to treated Alp1.sup.−/− mice (Table 15). In contrast, neither frontal nor parietal bones of treated Alp1.sup.−/− mice were significantly different from those of wild type mice. Adult skulls of LVL-RecAP16-treated Alp1.sup.−/− mice did not appear different from WT in terms of size and shape (
(232) TABLE-US-00015 TABLE 15 μCT analyses of cranial bones. Frontal and parietal bones were analzyed in WT, untreated Alpl.sup.−/− (vehicle), and RecAP16-treated Alpl.sup.−/− mice at p21. Values are reported as means ± SD. Bone Bone Tissue Tissue Bone Mineral Mineral Mineral Mineral Volume Content Density Content Density Fraction (mg) (mg/cc) (mg) (mg/cc) FRONTAL Alpl.sup.−/− .sup. 0.41 ± 0.06 * .sup. 0.008 ± 0.001 * .sup. 464 ± 27 * .sup. 0.004 ± 0.001 * .sup. 595 ± 28 * LVL- 0.72 ± 0.15 0.018 ± 0.005 615 ± 84 0.015 ± 0.006 697 ± 57 RecAP16 WT 0.70 ± 0.12 0.019 ± 0.008 615 ± 79 0.016 ± 0.008 709 ± 56 PARIETAL Alpl.sup.−/− .sup. 0.52 ± 0.05 * .sup. 0.018 ± 0.001 * .sup. 494 ± 21 * .sup. 0.005 ± 0.001 * .sup. 597 ± 12 * LVL- 0.72 ± 0.08 0.018 ± 0.007 608 ± 74 0.015 ± 0.006 691 ± 61 RecAP16 WT 0.76 ± 0.10 0.020 ± 0.007 645 ± 73 0.017 ± 0.007 726 ± 51 * Indicates statistical significance between genotypes and between treatment cohorts.
LVL-RecAP Treatment Partially Rescues Alp1.sup.−/− Dentoalveolar Defects
(233) Ablation of Alp1 in mice results in developmental mineralization defects in cementum, dentin, alveolar bone, and enamel (Foster et al., 2014a; Foster et al., 2014b; McKee et al. 2011; Yadav et al., 2012), consistent with case reports on human subjects with HPP. Absence of acellular cementum results in loss of periodontal attachment to the tooth root surface and premature tooth exfoliation, a hallmark of HPP. LVL-RecAP8-treated and vehicle-treated Alp1.sup.−/− mice were compared to WT at p25-26, when molar tooth formation is near completion. Radiography and μCT imaging revealed that, compared to controls (
(234) The LVL-RecAP16 cohort was compared with WT at p50-53 to determine effects on mature tooth structure and function. Radiography and μCT imaging indicated reduced alveolar and interproximal bone mineralization around molar teeth in Alp1.sup.−/− mouse mandibles, compared to controls (
(235) By histology, LVL-RecAP16 mice featured a mix of mineralized alveolar bone and osteoid, and a reduced but maintained PDL space (
(236) TABLE-US-00016 TABLE 16 μCT analyses of dentoalveolar tissues. First mandibular molars and associated alveolar bone were compared at p50-53 in WT (n = 5) and Alpl.sup.−/− mice treated with RecAP16 (n = 4). Values are reported as means ± SD. TV BV BV/TV TMD Thickness (mm.sup.3) (mm.sup.3) (%) (g HA/cm.sup.3) (μm) Enamel WT 0.25 ± 0.03 0.24 ± 0.02.sup. 98.77 ± 0.52 1.69 ± 0.05 70.2 ± 0.5 LVL- .sup. 0.20 ± 0.02 * 0.20 ± 0.02 * 98.97 ± 0.27 1.68 ± 0.03 63.0 ± 0.5 RecAP16 Crown WT 0.61 ± 0.04 0.56 ± 0.03.sup. 91.06 ± 0.64 ND ND LVL- .sup. 0.53 ± 0.03 * 0.46 ± 0.03 * .sup. 86.92 ± 0.69 * ND ND RecAP16 Root WT 0.65 ± 0.05 0.56 ± 0.05.sup. 85.84 ± 0.74 1.07 ± 0.01 140.4 ± 8.4 LVP- 0.56 ± 0.06 0.44 ± 0.04 * .sup. 77.27 ± 2.37 * .sup. 1.02 ± 0.01 * 121.5 ± 1.7 * RecAP16 Alveolar Bone WT 0.44 ± 0.04 0.29 ± 0.05.sup. 65.72 ± 5.08 1.01 ± 0.02 ND LVL- 0.32 ± 0.05* 0.15 ± 0.02 * .sup. 47.91 ± 4.25 * .sup. 0.88 ± 0.02 * ND RecAP16 * p < 0.05 by independent samples t-test ND = Not determined
LITERATURE REFERENCES TO EXAMPLE 8
(237) Anderson H. C., Sipe J. B., Hessle L., Dhanyamraju R., Atti E., Camacho N. P., Millán J. L. Impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice. Am. J. Pathol. 2004; 164(3):841-847. Bouxsein M L, Boyd S K, Christiansen B A, Guldberg R E, Jepsen K J and Müller R. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res 2010; 25(7):1468-86. Fedde K N, Blair L, Silverstein, J, Coburn S P, Ryan L M, Weinstein R S, Waymire K, Narisawa S, Millán, J L, MacGregor GR, Whyte M P, Alkaline phosphatase knockout mice recapitulate the metabolic and skeletal defects of infantile hypophosphatasia. J Bone Miner Res 1999; 14: 2015-2026. Foster B L, Nagatomo K J, Nociti F H, Fong H, Dunn D, Tran A B, Wang W, Narisawa S, Millán J L, Somerman M J 2012 Central role of pyrophosphate in acellular cementum formation. PLoS One 7(6):e38393. Foster B. L., Nagatomo K. J., Tso H. W., Tran A. B., Nociti F. H., Jr., Narisawa S., Yadav M. C., McKee M. D., Millan J. I., Somerman M. J. Tooth root dentin mineralization defects in a mouse model of hypophosphatasia. J Bone Miner Res. 2013; 28(2):271-82. Foster B L, Nociti F H, Jr., Somerman M J (2014a). The rachitic tooth. Endocr Rev 35(1):1-34. Foster B L, Ramnitz M S, Gafni R I, Burke A B, Boyce A M, Lee J S et al. (2014b). Rare Bone Diseases and Their Dental, Oral, and Craniofacial Manifestations. J Dent Res 93(7 suppl).7S-19S. Hessle L., Johnson K. A., Anderson H. C., Narisawa S., Sali A., Goding J. W., Terkeltaub R., Millán J. L. Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization. Proc. Natl. Acad. Sci. U.S.A. 2002; 99(14):9445-9449. Huesa C., Yadav M. C., Finnilá M. A., Goodyear S. R., Robins S. P., Tanner K. E., Aspden R. M., Millán J. L., Farquharson C. PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures. Bone. 2011; 48(5):1066-1074. Liu J, Nam H K, Campbell C, Gasque K C, Millán J L, Hatch N E. Tissue-nonspecific alkaline phosphatase deficiency causes abnormal craniofacial bone development in the Alp1(−/−) mouse model of infantile hypophosphatasia. Bone 2014; 67:81-94. McKee M. D., Nakano Y., Masica D. L., Gray J. J., Lemire I., Heft R., Whyte M. P., Crine P., Millán J. L. Enzyme replacement therapy prevents dental defects in a model of hypophosphatasia. J. Dent. Res. 2011; 90(4):470-476. Meganck J A, Kozloff K M, Thornton M M, Broski S M and Goldstein S A. Beam hardening artifacts in micro-computed tomography scanning can be reduced by X-ray beam filtration and the resulting images can be used to accurately measure BMD. Bone 2009; 45(6):1104-1116. Millán J L, Narisawa S, Lemire I, Loisel T P, Boileau G, Leonard P, Gramatikova S, Terkeltaub R, Pleshko Camacho N, McKee M D, Crine P and Whyte M P, Enzyme replacement therapy for murine hypophosphatasia. J Bone Miner Res 2008; 23: 777-787. Narisawa S, Wennberg C. Millán J L, Abnormal vitamin B6 metabolism in alkaline phosphatase knock-out mice causes multiple abnormalities, but not the impaired bone mineralization. J Pathol 2001; 193: 125-133. Narisawa S, Fröhlander N, Millán J L, Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia. Dev Dyn 1997; 208: 432-446. Umoh J U, Sampaio A V, Welch I, Pitelka V, Goldberg H A, Underhill T M et al. In vivo micro-CT analysis of bone remodeling in a rat calvarial defect model. Phys Med Biol 2009; 54(7):2147-61. Yadav M. C., Lemire I., Leonard P., Boileau G., Blond L., Beliveau M., Cory E., Sah R. L., Whyte M. P., Crine P., Millán J. L. Dose response of bone-targeted enzyme replacement for murine hypophosphatasia. Bone. 2011; 49(2):250-256. Yadav M. C., de Oliveira R. C., Foster B. L., Fong H., Cory E., Narisawa S., Sah R. L., Somerman M., Whyte M. P., Millán J. L. Enzyme replacement prevents enamel defects in hypophosphatasia mice. J. Bone Miner. Res. 2012; 27(8):1722-1734. Yadav, M. C., Huesa, C., Narisawa, S., Hoylaerts, M. F., Moreau, A., Farquharson, C. and Millán, J. L. Ablation of osteopontin improves the skeletal phenotype of Phospho1.sup.−/− mice. J. Bone Miner. Res. In Press (2014).
Example 9
(238) Alkaline Phosphatase Protects Against Renal Inflammation
(239) Methods
(240) Cell Culture
(241) Routinely, ciPTEC were cultured at 33° C. (Wilmer, 2010). Cells were transfected with Simian Virus 40 T-antigen and the essential catalytic subunit of human telomerase, allowing them to constantly proliferate. Cells were cultured in DMEM/Ham's F-12, phenolred-free (Gibco, Paisly, United Kingdom), supplemented with ITS (5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium; Sigma-Aldrich, Zwijndrecht, The Netherlands), 36 ng/ml hydrocortisone (Sig-ma-Aldrich), 10 ng/ml epidermal growth factor (Sigma-Aldrich), 40 μg/ml tri-iodothyronine and 10% fetal calf serum (Greiner Bio-One, Kremsmünster, Austria). Preceding an experiment, cells were seeded in a well-plate (48400 cells/cm.sup.2), incubated for 1 day at 33° C. followed by a 7-day maturation period at 37° C. On the day of the experiment, cells were pre-incubated for two hrs with LVL-RecAP (1, 5 or 10 U/ml, kind gift from AM-Pharma, Bunnik, The Netherlands) (Kiffer-Moreira, 2014), followed by incubation for 24 hrs with 10 μg/ml LPS (E. coli 0127:B8; Sigma-Aldrich) dissolved in 10 mM HEPES HBSS, pH 7.4 (HEPES: Roche Diagnostics, Mannheim, Germany; HBSS: Gibco). Alternatively, 10 U/ml LVL-RecAP (˜17 μg/ml) was administered to LPS-incubated cells simultaneously or after two hrs. Control cells were incubated with culture medium solely. DLPS (E. coli 055:B5; Sigma-Aldrich, 10 μg/ml) and inactive LVL-RecAP (17 μg/ml, kind gift from AM-Pharma) were used as negative controls. In different sets of experiments, LPS was substituted for human TNF-α recombinant protein (Ebioscience, Vienna, Austria), or supernatant of PBMCs, prestimulated for 24 hrs with or without LPS (1 ng/ml). All experiments (n=5) were minimally performed in duplicate.
(242) Isolation of Peripheral Blood Mononuclear Cells
(243) PBMCs were isolated from buffy coats obtained from healthy blood donors (blood bank Nijmegen, n=5) by differential centrifugation over Ficoll-Pague Plus (GE Healthcare, Diegem, Belgium). PBMCs were resuspended in RPMI-1640 medium (Gibco) enriched with 0.5 mg/ml gentamicin (Sigma-Aldrich), 1 mM pyruvate (Gibco) and 2 mM glutamax (Gibco). Cells were seeded in 96-well plates at a density of 0.5×10.sup.6 cells/well, pre-incubated with or without AP (10 units/ml) for 2 hrs, followed by LPS incubation (1 ng/ml) for 24 hrs. All experiments were performed in duplicate.
(244) ATP Measurement and Cell Viability Assay
(245) Supernatant was collected 30 minutes after LPS administration, with or without LVL-RecAP-pretreatment, followed by direct measurement of ATP production using the ATP Bioluminescence Assay Kit CLS II (Roche Diagnostics) according to manufacturer's protocol. Cell viability was assessed after 24 hrs of LPS incubation by performing the MTT assay. In short, medium was substituted for 100 μl prewarmed MTT-solution (Sigma-Aldrich; 0.5 mg/ml in culture medium), incubated for 3 hrs at 37° C., followed by the addition of 200 μl DMSO to solubilize intracellular precipitated formazan crystals. Dye extinction was measured at 570 nm with wavelength correction of 670 nm.
(246) Animal Model
(247) Animal experiments were performed according to the National Institutes of Health guidelines and protocols were approved by the institutional review board for animal experiments. Male specific-pathogen free Sprague-Dawley rats (RjHan:SD: Janvier, France) were divided into three groups: placebo (n=6), LPS (n=6) or LPS+LVL-RecAP (n=6). A baseline plasma sample (lithium-heparin blood) was collected seven days preceding the experiment through a tail vein puncture using a Multivette (Sarstedt, Etten-Leur, the Netherlands). Three days preceding the experiment, the baseline renal function was assed as FITC-sinistrin t.sub.1/2 (Schock-Kusch, 2011). At t=0 hrs, placebo (0.9% NaCl, saline) or 0.3 mg/kg BW LPS (E. coli 0127:B8, dissolved in saline) was administered as an IV bolus to induce LPS-induced renal failure. At t=1.5 hrs plasma was obtained as described above. At t=2 hrs, rat received an IV bolus of placebo or LVL-RecAP (1000 U/kg BW, diluted in saline) followed by a second measurement of renal function. At t=5 hrs, all animals received 5 ml saline (s.c.) to prevent dehydration, followed by a 16 hour urine collection period. At t=21.5 hrs the third transcutaneous measurement was performed. At t=24 hrs, rats were anesthetized (i.p., 3 mg/kg BW xylazine and 80 mg/kg BW ketamine 10%), a retrobulbar lithium-heparin blood sample was withdrawn to obtain plasma, and whole body perfusion was started (6 min, saline+50 IU/ml heparin, 210 mbar; 3 min, 4% paraformaldehyde (PFA; 210 mbar). After saline perfusion, the right kidney was carefully removed, snap frozen and stored at −80° C. until processing. The left kidney, removed after PFA perfusion, was stored in 4% PFA at 4° C. until processed for histology and immunohistochemistry. One animal from the LPS+LVL-RecAP group and one urine sample from the placebo group were excluded due to injection and collection difficulties, respectively.
(248) Renal Function Measurements
(249) Renal function was assessed in freely moving awake rats through transcutaneously measured elimination kinetics of FITC-sinistrin (Fresenius Kabi, Linz, Austria), a commercially available marker of GFR, by using a novel measurement device as published before (Schock-Kusch 2011, Schock-Kusch 2009). Briefly, rats were anesthetized by isoflurane inhalation (5% induction, 1.5-2% maintenance; Abbott Laboratories, Ill., USA) and shaved on the back. The optical part of the device was fixed on this depilated region using a specifically designed double-sided adhesive patch (Lohmann GmbH, Neuwied, Germany), whereas the electronic part of the device was incorporated into a rodent jacket (Lomir Biomedical, Malone, USA). After establishing the baseline signal, FITC-sinistrin (5 mg per 100 g BW, diluted in buffered saline) was injected in the tail vein. Thereafter, the animals were allowed to recover from anesthesia while the measurement continued for approximately 120 minutes post-injection. T.sub.1/2 was calculated by a one-compartment model applied on the transcutaneously measured FITC-sinistrin elimination kinetics (Schock-Kusch, 2009). In addition, parameters of renal function were determined in plasma and urine samples using the Hitachi 704 automatic analyzer (Boehringer Mannheim, Mannheim, Germany). Fractional urea excretion and endogenous creatinine clearance were calculated with average plasma values of t=1.5 and t=24.
(250) Histology and Immunohistochemistry
(251) After fixation for at least 24 hrs, tissue was processed, embedded in paraplast and sectioned at 3 μM thickness. For routine histology, HE staining was performed on renal tissue. Renal injury was assessed using a scoring system with a scale from 0 to 4 (0=no changes; 4=severe damage e.g. marked tubule cell changes). KIM-1 was detected by primary antibody goat-anti-rat KIM-1 (1:50; AF3689, R&D Systems, Abingdon, UK) and secondary antibody rabbit-anti-goat IgG (1:200; P0449, DAKO, Heverlee, Belgium). Immunostaining was visualized with VECTASTAIN Eline ABC system reagents (Vector Labs, Amsterdam, Netherlands) and 3,3′-Diaminobenzidine (DAB, Sigma-Aldrich), followed by haematoxyline counterstain. All scoring was performed in a blinded fashion.
(252) Cytokines and Renal Injury Markers
(253) Human ELISA kits (R&D Systems) were used to determine TNF-α, IL-6 and IL-8 in supernatant according to manufacturer's instructions. Plasma cytokine levels (IL-1ß, IL-6, IL-10, TNF-α, INF-γ) were determined by a simultaneous Luminex assay according to the manufacturer's instructions (Millipore, Cork, Ireland). KIM-1 and NGAL were determined by ELISA (R&D Systems) according to manufacturer's instructions.
(254) Tissue Homogenization
(255) Snap frozen kidneys were homogenized by the TissueLyser LT (Qiagen, Venlo, The Netherlands) according to manufacturer's instructions, in Tissue Protein Extraction Reagent (T-PER; Thermo Scientific, Rockford, USA), supplemented with complete EDTA-free protease inhibitor cocktail tablets (Roche Applied Science, Almere, The Netherlands). Total protein content was determined using the bicinchonicic acid protein assay kit (Thermo Scientific) and samples were stored at −80° C. until assayed.
(256) Real-Time PCR Analyses
(257) RNA was extracted from frozen cell pellet or pulverized kidneys (2000, 30 sec; Mikro-dismembrator U, Sartorius Stedim Biotech, Aubagne Cedex, France) by Trizol reagent. RNA was reverse-transcribed into cDNA using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase (Invitrogen, Breda, The Netherlands). Real-time quantitative PCR (RQ-PCR) was performed using Taqman® (Applied Biosystems, Carlsbad, USA). Genes were amplified and normalized to the expression of GAPDH (ciPTEC: Ct: 18.9±0.1; renal tissue: Ct: 24.8±0.2). The PCR reaction started with a 2 min incubation step at 50° C. followed by initial denaturation for 10 min at 95° C., and 40 cycles of 15 sec at 95° C. and 1 min at 60° C. Differences between groups were calculated by the comparative ΔΔCt method. Primers/probe sets are summarized in Table 17.
(258) Urinary Purine Content
(259) Urinary adenosine, AMP, ADP, ATP and cAMP content was determined by HPLC. In brief, 4 volumes of urine were mixed with 1 volume of chloroacetaldehyde (6× diluted in 1M acetate buffer, pH 4.5; Sigma-Aldrich), followed by derivatization (60 min, 70° C., 500 rpm) and centrifugation (3 min, RT, 13400 rpm), whereafter the supernatant was transferred to a HPLC vial and injected. Purines were separated by HPLC system (Thermo Scientific) using a Polaris C18-A column (150×4.6 mm) with gradient elution using eluent A (0.1M K.sub.2HPO.sub.4, 10 mM TBAHS (pH 6.5), and 2% MeOH) and eluent B (H.sub.2O: ACN: THF; 50:49:1). Retention times were 7.1 (adenosine), 8.4 (AMP), 12.5 (ADP), 16.2 (ATP) and 14.8 min (cAMP). Quantification was based on peak areas of the samples and reference standards measured with fluorescence (excitation: 280 nm; emission: 420 nm).
(260) Statistical Analysis
(261) Data are expressed as mean±SEM or median [25th percentile, 75.sup.th percentile]. Normality of data was assessed by Kolmogorov-Smirnov test. Statistical differences between groups were estimated by ANOVA with post-hoc comparisons using Bonferroni's multiple comparison test or by Kruskal-Wallis test with Dunn's post-test. A two-sided p-value less than 0.05 was considered statistically significant. All tests were performed with Graphpad Prism 5.00 for Windows (Graphpad Software Inc. San Diego, Calif., USA).
(262) LVL-RecAP Attenuates the LPS-Induced Inflammatory Response In Vitro
(263) Pre-treatment of human ciPTEC (Wilmer, 2010) with LVL-RecAP dose-dependently attenuated the LPS-induced cytokine production of TNF-α, IL-6 and IL-8 on the gene and protein level (
(264) The In Vitro Effects of LVL-RecAP are Renal Specific and not Restricted to LPS-Induced Inflammation
(265) To investigate further the renal protective mechanism of LVL-RecAP, ciPTEC were incubated with the pro-inflammatory cytokine TNF-α, which cannot be dephosphorylated by calf IAP (Chen, 2010). The TNF-α induced cytokine production of IL-6 and IL-8 was also attenuated by LVL-RecAP pretreatment, whereas inactive LVL-RecAP had no effect (
(266) In the pathogenesis of sepsis-associated AKI, LPS induces a local inflammatory response through binding to TLR4 expressed on PTEC (ciPTEC Ct: 30.5±3.9; n=5). Another hallmark of the disease is the systemic inflammatory response, which affects both renal epithelial and endothelial cells causing the development of AKI (Peters, 2014). To mimic this endotoxin-induced renal inflammation, ciPTEC were incubated with the supernatant of LPS-stimulated peripheral blood mononuclear cells (PBMCs, 1 ng/ml LPS). This induced the production of IL-6 and IL-8, which was decreased when ciPTEC were pretreated with LVL-RecAP (
(267) LVL-RecAP May Exert Renal Protective In Vitro Effects Through the ATP/Adenosine Pathway
(268) A second potential target of LVL-RecAP is ATP, released during cell stress caused by e.g. inflammation and hypoxia (Eltzschig, 2012). Extracellular ATP has detrimental effects, but can be converted by ectonucleotidases (e.g. AP) into ADP, AMP and eventually into adenosine, exerting anti-inflammatory and tissue-protective effects through binding to one of the adenosine receptors A1, A2A, A2B and A3 (Bauerle, 2011, Di Sole, 2008). Interestingly, while the adenosine receptors A1, A2B and A3 expression in ciPTEC were not affected by LPS incubation (data not shown), the A2A expression was up-regulated upon LPS stimulation (fold increase: 4.1±0.4; p<0.001 compared to placebo). This effect was attenuated by LVL-RecAP co-treatment (fold increase: 2.9±0.2; p<0.001 compared to placebo; p<0.05 compared to LPS), suggesting a role of the adenosine pathway in the protective effect of LVL-RecAP. Furthermore, we observed increased extracellular ATP concentrations following LPS incubation, which was more pronounced with a higher LPS concentration but reversed by LVL-RecAP preincubation (
(269) LVL-RecAP Treatment During LPS-Induced AKI in Rats Attenuates Impaired Renal Function
(270) To confirm the beneficial effects of LVL-RecAP in vivo, AKI was induced in rats by LPS (0.3 mg/kg BW) and renal function was assessed by the transcutaneous measurement of the fluorescein isothiocyanate (FITC)-labeled sinistrin kinetics as previously reported (Schock-Kusch, 2011). FITC-sinistrin is cleared by the kidneys through filtration solely and its disappearance from the plasma compartment can be measured transcutaneously in real-time (Schock-Kusch, 2011, Schock-Kusch, 2009). This allows investigating the progression of AKI in a more accurate manner as compared to the commonly used creatinine clearance. Preceding LPS injection, a baseline blood sample was drawn to determine clinical parameters and plasma cytokines, and the baseline FITC-sinistrin half-life (t.sub.1/2) was determined from the measured kinetics to ascertain homogeneity between groups (data not shown). After 1.5 hrs, LPS treatment resulted in increased plasma cytokines levels, abnormalities in several plasma parameters (Table 18), piloerection, diarrhea and reduced spontaneous activity, confirming the presence of systemic inflammation. Two hrs after LPS administration, rats were treated with LVL-RecAP (1000 U/kg BW) or placebo (saline), directly followed by transcutaneous renal function measurements. LPS significantly prolonged FITC-sinistrin t.sub.1/2, revealing a significant reduction in renal function. This trend was attenuated by LVL-RecAP treatment (
(271) LVL-RecAP Prevents Renal Injury During LPS-Induced AKI In Vivo
(272) The renal protective effect of LVL-RecAP on LPS-induced AKI was investigated further through evaluation of renal histology and specific tubular injury markers. No differences in histology were found between the treatment groups, with changes that ranged from no damage (0) till minimal degenerative changes like foamy appearance and minimal swelling of proximal tubular cells (1) and foamy appearance and moderate swelling as well as a few cases of apoptosis (2) (Placebo; 1 [0.75-2]; LPS: 1.5 [0-2]; LPS+LVL-RecAP: 1 [0-1.0]). LPS treatment resulted in a significant increase in renal IL-6 expression levels, while other cytokines and injury markers (MPO, myeloperoxidase; BAX, Bcl2-associated X protein; iNOS, inducible nitric oxide synthase) were not affected (Table 20). LVL-RecAP could not reduce renal IL-6 expression levels, but did enhance renal expression of the anti-inflammatory cytokine IL-10 (Table 20). Furthermore, LPS administration resulted in a significant increase in the urinary excretion of kidney injury molecule (KIM)-1 and neutrophil gelatinase-associated lipocalin (NGAL), which was accompanied by increased renal gene expression levels. This effect was prevented by LVL-RecAP co-administration (
(273) Reduced Urinary Adenosine Excretion During LPS-Induced AKI In Vivo
(274) In order to elucidate further the renal protective mechanism of LVL-RecAP, we investigated the role of the ATP-adenosine pathway. LPS treatment tended to reduce the gene expression levels in the kidney for all four adenosine receptors, of which only adenosine receptor A3 reached statistical significance (Table 20). Interestingly, LPS treatment significantly decreased the urinary excretion of adenosine (placebo: 68.0±7.8 pg adenosine/10 μg creatinine; LPS: 19.4±6.3 pg adenosine/10 μg creatinine; p<0.001), without altering the excretion of cAMP, ATP, ADP and AMP (data not shown). This may suggest that the kidney utilizes adenosine during LPS-induced AKI. LVL-RecAP treatment had no effect on adenosine receptor gene expression (Table 20), or on urinary adenosine excretion (LPS+LVL-RecAP: 16.7±6.8 μg adenosine/10 μg creatinine: p<0.001 compared to placebo) compared to LPS alone.
(275) Tables
(276) TABLE-US-00017 TABLE 17 Primer/probe specifications Gene symbol Gene name Assay ID ciPTEC GAPDH glyceraldehyde-3-phosphate Hs02758991_g1 dehydrogenase TNF-α tumor necrosis factor Hs01113624_g1 IL-6 interleukin 6 Hs00985639_m1 IL-8 interleukin 8 Hs00174103_m1 TLR4 toll-like receptor 4 Hs00152939_m1 ADORA1 adenosine A1 receptor Hs00379752_m1 ADORA2A adenosine A2a receptor Hs00169123_m1 ADORA2B adenosine A2b receptor Hs00386497_m1 ADORA3 adenosine A3 receptor Hs01560269_m1 Rat kidney GAPDH glyceraldehyde-3-phosphate Rn01775763_g1 dehydrogenase IL-1β interleukin 1 beta Rn00580432_m1 IL-6 interleukin 6 Rn01410330_m1 IL-10 interleukin 10 Rn00563409_m1 TNF-α tumor necrosis factor Rn99999017_m1 IFN-γ interferon gamma Rn00594078_m1 HAVCR1 hepatitis A virus cellular receptor 1 Rn00597703_m1 LCN2 lipocalin 2 Rn00590612_m1 MPO myeloperoxidase Rn01460204_m1 BAX Bcl2-associated X protein Rn02532082_g1 NOS2 nitric oxide synthase 2, inducible Rn00561646_m1 ADORA1 adenosine A1 receptor Rn00567668_m1 ADORA2A adenosine A2a receptor Rn00583935_m1 ADORA2B adenosine A2b receptor Rn00567697_m1 ADORA3 adenosine A3 receptor Rn00563680_m1
(277) TABLE-US-00018 TABLE 18 Plasma cytokines and experimental parameters Placebo LPS LPS + recAP Cytokines IL-1β (pg/ml) 9 ± 9 6913 ± 1362.sup.# 5764 ± 1259.sup.# IL-6 (pg/ml) ND 74294 ± 11240.sup.# 70247 ± 13812.sup.# IL-10 (pg/ml) 17 ± 17 10054 ± 2017.sup.# 8693 ± 2513.sup.# TNF-α (pg/ml) 1 ± 1 21071 ± 3375.sup.# 10514 ± 889.sup.# *.sup. INF-γ (pg/ml) ND ND ND Plasma parameters Creatinine 0.16 ± 0.01 .sup. 0.30 ± 0.05 0.25 ± 0.04.sup. (mg/dl) Urea (mg/dl) 28 ± 2 39 ± 2.sup.# 48 ± 3.sup.# * Lactate (mg/dl) 24 ± 5 34 ± 2.sup. 33 ± 3 .sup. Glucose 143 ± 3 260 ± 25.sup.# 216 ± 13.sup.# (mg/dl) Protein 58 [56-58].sup. 58 [48-61].sup. 56 [56-58].sup. (mg/ml) Calcium 2.59 ± 0.03 2.43 ± 0.03.sup.# 2.41 ± 0.06.sup.# (mmol/l) Inorganic 3.01 ± 0.10 .sup. 2.76 ± 0.09 2.81 ± 0.10.sup. Phosphorus (mmol/l) Sodium 149 ± 2 148 ± 2 .sup. 146 ± 3 .sup. (mmol/l) Potassium 5.89 [5.66-6.21] 4.87 [4.66-5.23] 5.08 [4.76-5.18] (mmol/l) Plasma parameters were determined 1.5 hrs after LPS administration. Data is expressed as mean ± SEM, and median [25.sup.th percentile, 75.sup.th percentile], depending on the distribution of each parameter. Differences in distribution of plasma parameters compared to t = 24 is likely to be related to sample size. Significant differences estimated using one-way ANOVA with Bonferroni post-test, or Kruskal-Wallis test with Dunns post-test. Placebo, LPS n = 6; LPS + recAP n = 5. .sup.#p < 0.05 compared to placebo. * p < 0.05 compared to LPS. LPS, Lipopolysaccharide; recAP, recombinant Alkaline Phosphatase; ND: not detected.
(278) TABLE-US-00019 TABLE 19 Plasma and urinary parameters Placebo LPS LPS + recAP Plasma parameters Creatinine 0.20 ± 0.01 0.24 ± 0.02 0.20 ± 0.01 (mg/dl) Urea (mg/dl) 28 ± 2 48 ± 4.sup.#.sup. 36 ± 2* Lactate (mg/dl) 10 [8-31] 14 [12-21] 12.0 [10-14] Glucose 144 ± 7 132 ± 11 161 ± 3 (mg/dl) Protein 56 [54-57] 56 [52-57] 54 [54-58] (mg/ml) Calcium 2.84 [2.7-3.1] 2.65 [2.6-2.7] 2.70 [2.6-2.8] (mmol/l) Inorganic 2.95 ± 0.09 3.06 ± 0.10 2.95 ± 0.09 Phosphorus (mmol/l) Sodium 150 [148-157] 154 [147-155] 150 [141-154] (mmol/l) Potassium 4.24 ± 0.04 4.32 ± 0.10 4.42 ± 0.24 (mmol/l) Urinary parameters Creatinine (mg) 5.4 ± 0.3 6.1 ± 0.3 5.7 ± 0.6 Urea (mg) 267 ± 26 482 ± 21.sup.#.sup. 440 ± 19.sup.#.sup. Albumin (μg) 0 [0-54] 0 [0-663] 310 [0-419] Glucose (mg) 1.59 ± 0.25 1.74 ± 0.28 0.99 ± 0.27 Protein (μg) 38 ± 6 67 ± 9 61 ± 9 Calcium 4.1 ± 0.7 7.2 ± 0.9 7.4 ± 1.7 (μmol) Inorganic 0.50 [0.3-0.9] 0.63 [0.6-1.0] 0.64 [0.6-0.8] Phosphorus (mmol) Sodium 1.6 [1.3-2.0] 1.5 [1.3-1.7] 1.5 [1.4-2.4] (mmol) Potassium 1.6 ± 0.3 .sup. 2.3 ± 0.4.sup.# .sup. 1.9 ± 0.1.sup.# (mmol) Plasma parameters were determined 24 hrs after LPS administration. Urinary parameters were determined between 5 and 21 hrs after LPS administration. Data is expressed as mean ± SEM, and median [25.sup.th percentile, 75.sup.th percentile], depending on the distribution of each parameter. Significant differences estimated using Kruskal-Wallis test with Dunns post-test or one-way ANOVA with Bonferroni post-test. Placebo, LPS n = 6; LPS + recAP n = 5; urinary parameters: Placebo n = 5. .sup.#p < 0.05 compared to placebo. *p < 0.05 compared to LPS. LPS, Lipopolysaccharide; recAP, recombinant Alkaline Phosphatase.
(279) TABLE-US-00020 TABLE 20 Renal gene expression levels Ct values Fold increase (2{circumflex over ( )}ΔΔCt) Placebo LPS LPS + recAP Placebo LPS LPS + recAP Cytokines IL-1β 27.7 ± 0.5 26.6 ± 0.2 26.9 ± 0.3 1.1 ± 0.2 1.6 ± 0.5 1.7 ± 0.5 IL-6 37.4 [36.3-38.0] 33.5 [33.3-34.8] 34.7 [34.4-36.4] 1.2 ± 0.3 .sup. 4.9 ± 0.5.sup.# .sup. 5.2 ± 1.2.sup.# IL-10 34.8 ± 0.4 32.5 ± 0.1 32.9 ± 0.3 1.1 ± 0.2 3.3 ± 0.9 .sup. 5.5 ± 1.2.sup.# TNF-α 36.7 ± 0.2 35.8 ± 0.4 37.2 ± 0.5 1.1 ± 0.2 1.0 ± 0.1 1.0 ± 0.3 INF-γ 36.3 ± 0.3 35.3 ± 0.4 35.6 ± 0.2 1.1 ± 0.2 1.3 ± 0.3 1.7 ± 0.2 Injury markers KIM-1 31.7 ± 0.4 22.9 ± 0.7 25.3 ± 0.6 0.8 [0.6-1.9] .sup. 430 [195-530].sup.# 113 [43-336].sup. NGAL 29.1 ± 0.2 24.1 ± 1.4 25.5 ± 1.6 1.2 ± 0.3 33 ± 9.sup.# 28 ± 8 MPO 32.4 ± 0.3 31.4 ± 0.7 33.0 ± 1.0 1.0 [0.4-2.9] 1.4 [0.4-3.9] 0.6 [0.3-3.2] BAX 25.2 ± 0.2 24.8 ± 0.1 25.1 ± 0.3 0.9 [0.7-1.5] 0.7 [0.3-2.1] 1.0 [0.5-1.3] iNOS 36.2 ± 0.5 35.0 ± 0.2 34.8 ± 0.8 1.1 ± 0.3 1.8 ± 0.5 2.6 ± 0.8 Adenosine receptors A1 28.6 ± 0.3 27.9 ± 0.3 29.3 ± 0.6 1.3 [0.4-2.3] 0.7 [0.5-1.4] 0.8 [0.3-1.5] A2A 27.0 ± 0.1 26.4 ± 0.2 27.3 ± 0.3 1.3 ± 0.4 0.8 ± 0.2 0.9 ± 0.2 A2B 29.0 [28.9-29.3] 28.4 [28.1-29.0] 29.0 [28.9-30.2] 1.2 ± 0.3 0.8 ± 0.2 0.8 ± 0.1 A3 34.3 ± 0.3 34.2 ± 0.4 35.0 ± 0.6 1.0 ± 0.1 .sup. 0.5 ± 0.1.sup.# 0.7 ± 0.1 Data is expressed as mean ± SEM, and median [25.sup.th percentile, 75.sup.th percentile], depending on the distribution of each parameter. Significant differences of the fold increase was estimated using Kruskal-Wallis test with Dunns post-test or one-way ANOVA with Bonferroni post-test. Placebo, LPS n = 6; LPS + recAP n = 5. .sup.#p < 0.05 compared to placebo. * p < 0.05 compared to LPS. LPS, Lipopolysaccharide; recAP, recombinant Alkaline Phosphatase; MPO, myeloperoxidase; BAX, Bcl2-associated X protein; iNOS, inducible nitric oxide synthase.
LITERATURE REFERENCES TO EXAMPLE 9
(280) Bauerle, J. D., Grenz, A., Kim, J. H., Lee, H. T., and Eltzschig, H. K. 2011. Adenosine generation and signaling during acute kidney injury. J Am Soc Nephrol 22:14-20. Chen, K. T., Malo, M. S., Moss, A. K., Zeller, S., Johnson, P., Ebrahimi, F., Mostafa, G., Alam, S. N., Ramasamy, S., Warren, H. S., et al. 2010. Identification of specific targets for the gut mucosal defense factor intestinal alkaline phosphatase. Am J Physiol Gastrointest Liver Physiol 299:G467-475. Di Sole, F. 2008. Adenosine and renal tubular function. Curr Opin Nephrol Hypertens 17:399-407. Eltzschig, H. K., Sitkovsky, M. V., and Robson, S. C. 2012. Purinergic signaling during inflammation. N Engl J Med 367:2322-2333. Kiffer-Moreira, T., Sheen, C. R., Gasque, K. C., Bolean, M., Ciancaglini, P., van Elsas, A., Hoylaerts, M. F., and Milan, J. L. 2014. Catalytic signature of a heat-stable, chimeric human alkaline phosphatase with therapeutic potential. PLoS One 9:e89374. Peters, E., Heemskerk, S., Masereeuw, R., and Pickkers, P. 2014. Alkaline Phosphatase: A Possible Treatment for Sepsis-Associated Acute Kidney Injury in Critically Ill Patients. Am J Kidney Dis. 63:1038-48 Schock-Kusch, D., Sadick, M., Henninger, N., Kraenzlin, B., Claus, G., Kloetzer, H. M., Weiss, C., Pill, J., and Gretz, N. 2009. Transcutaneous measurement of glomerular filtration rate using FITC-sinistrin in rats. Nephrol Dial Transplant 24:2997-3001. Schock-Kusch, D., Xie, Q., Shulhevich, Y., Hesser, J., Stsepankou, D., Sadick, M., Koenig, S., Hoecklin, F., Pill, J., and Gretz, N. 2011. Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves. Kidney Int 79:1254-1258. Wilmer, M. J., Saleem, M. A., Masereeuw, R., Ni, L., van der Velden, T. J., Russel, F. G., Mathieson, P. W., Monnens, L. A., van den Heuvel, L. P., and Levtchenko, E. N. 2010. Novel conditionally immortalized human proximal tubule cell line expressing functional influx and efflux transporters. Cell Tissue Res 339:449-457.
Example 10
(281) Comparison LVL-RecAP with RecAP under different temperature conditions
(282) 10.6. Materials
(283) 10.6.1 Reference Standards
(284) LVL-RecAP
(285) Batch number: NB1963p1 PRA ID: 14-049 Protein content: 9.9 mg/mL (OD280) Activity: 6537 U/mL (660 U/mg) Storage condition: nominal at −70° C. Expiry date: 8 Jan. 2015
RecAP Batch number: 2013-052, lot 62 PRA ID: 14-311 Protein content: 13.3 mg/mL (OD280) Activity: 9871 U/mL (742 U/mg) Storage condition: at 2-8° C. Expiry date: 6 Jun. 2016
10.6.2 Blank Matrix
(286) The following biological matrix was used for preparation of sample solutions. Matrix: serum Species: human Supplier: Sera Laboratories International, Haywards Heath, UK Storage condition: At a nominal storage temperature −20° C. PRA IDs: 14-0624, 14-0647 and 14-0652 Expiry dates: 2 May 2016 (14-0624), 6 May 2016 (14-0647 and 14-0652)
10.7. Methods
10.7.1 Preparation of Solutions
10.7.1.1 2 M Sodium Hydroxide
(287) A 2 molar sodium hydroxide solution was prepared by dissolving 8 g of sodium hydroxide in approximately 90 mL Milli-Q water and after cooling to room temperature the volume was adjusted to 100 mL. The solution was stored at room temperature up to a maximum of one month.
(288) 10.7.1.2 1 M Magnesium Chloride
(289) A 1 molar magnesium chloride solution was prepared by dissolving 4.06 g magnesium chloride hexahydrate in approximately 16 mL Milli-Q water. After dissolving the volume was adjusted to 20 mL. The solution was stored at nominal +4° C. up to a maximum of one month.
(290) 10.7.1.3 0.1 M Zinc Chloride
(291) A 0.1 molar zinc chloride solution was prepared by dissolving 272.5 mg zinc chloride in approximately 16 mL Milli-Q water. After dissolving, the volume was adjusted to 20 mL.
(292) The solution was stored at nominal +4° C. up to a maximum of one month.
(293) 10.7.1.4 0.025M Glycine pH 9.6 Solution for 25° C. Method
(294) A 0.025 molar glycine pH 9.6 solution was prepared by dissolving 3.76 g of glycine in approximately 1800 mL Milli-Q water. The solution was warmed to 25° C. and adjusted to pH 9.6 with 2 M sodium hydroxide (see Section 10.7.1.1). The volume was made up to 2000 mL and the pH was rechecked. The pH should be pH 9.6 at 25° C. The solution was stored at nominal +4° C. up to a maximum of one week.
(295) 10.7.1.5 Enzyme Diluent Buffer for 25° C. Method
(296) The enzyme diluent buffer was prepared by mixing 0.5 mL 1 M magnesium chloride (see Section 10.7.1.2) with 0.5 mL 0.1 M zinc chloride (see Section 10.7.1.3) and 500 mL 0.025 M glycine pH 9.6 solution (see Section 10.7.1.4). To this solution, 5.00 g mannitol and 0.25 g bovine serum albumin was added and dissolved under stirring. The pH was checked and if deemed necessary adjusted to pH 9.6 at 25° C. using 2 M sodium hydroxide. The enzyme diluent buffer was prepared freshly every day.
(297) 10.7.1.6 0.0103 M p-Nitrophenyl Phosphate pH 9.6 for 25° C. Method
(298) A 0.0103 M p-nitrophenyl phosphate pH 9.6 was prepared by dissolving 1528 mg p-nitrophenyl phosphate in approximately 360 mL 0.025 M glycine pH 9.6 solution (see Section 10.7.1.4). The pH was checked and if deemed necessary adjusted to pH 9.6 at 25° C. using 2 M sodium hydroxide (see Section 10.7.1.1). After checking the pH, the volume was adjusted to 400 mL with 0.025 M glycine pH 9.6 solution. The solution was stored at nominal +4° C. up to a maximum of 5 days.
(299) 10.7.1.7 Working Substrate for 25° C. Method
(300) Working substrate was prepared by mixing 120 mL 0.0103 M p-nitrophenyl phosphate pH 9.6 solution (see Section 10.7.1.6) with 1.25 mL 1 M magnesium chloride solution (see Section 10.7.1.2). To this solution approximately 15 mL 0.025 M glycine pH 9.6 solution (see Section 10.7.1.4) was added and the pH was checked and if deemed necessary adjusted to pH 9.6 at 25° C. using 2 M sodium hydroxide (see Section 10.7.1.1). The volume was adjusted to 145 mL with 0.025 M glycine pH 9.6 solution. Working substrate was prepared freshly every day.
(301) 10.7.1.8 0.025M Glycine pH 9.6 Solution for 37° C. Method
(302) A 0.025 molar glycine pH 9.6 solution was prepared by dissolving 3.76 g of glycine in approximately 1800 mL Milli-Q water. The solution was warmed to 25° C. and adjusted to pH 9.6 with 2 M sodium hydroxide (see Section 10.7.1.1). The volume was made up to 2000 mL and the pH was rechecked. The pH should be pH 9.6 at 37° C. The solution was stored at nominal +4° C. up to a maximum of one week.
(303) 10.7.1.9 Enzyme Diluent Buffer for 37° C. Method
(304) The enzyme diluent buffer was prepared by mixing 0.5 mL 1 M magnesium chloride (see Section 10.7.1.2) with 0.5 mL 0.1 M zinc chloride (see Section 10.7.1.3) and 500 mL 0.025 M glycine pH 9.6 solution (see Section 10.7.1.8). To this solution, 5.00 g mannitol and 0.25 g bovine serum albumin was added and dissolved under stirring. The pH was checked and if deemed necessary adjusted to pH 9.6 at 37° C. using 2 M sodium hydroxide. The enzyme diluent buffer was prepared freshly every day.
(305) 10.7.1.10 0.0103 M p-Nitrophenyl Phosphate pH 9.6 for 37° C. Method
(306) A 0.0103 M p-nitrophenyl phosphate pH 9.6 was prepared by dissolving 1528 mg p-nitrophenyl phosphate in approximately 360 mL 0.025 M glycine pH 9.6 solution (see Section 10.7.1.8). The pH was checked and if deemed necessary adjusted to pH 9.6 at 37° C. using 2 M sodium hydroxide (see Section 10.7.1.1). After checking the pH, the volume was adjusted to 400 mL with 0.025 M glycine pH 9.6 solution. The solution was stored at nominal +4° C. up to a maximum of 5 days.
(307) 10.7.1.11 Working Substrate for 37° C. Method
(308) Working substrate was prepared by mixing 120 mL 0.0103 M p-nitrophenyl phosphate pH 9.6 solution (see Section 10.7.1.10) with 1.25 mL 1 M magnesium chloride solution (see Section 10.7.1.2). To this solution approximately 15 mL 0.025 M glycine pH 9.6 solution (see Section 10.7.1.4) was added and the pH was checked and if deemed necessary adjusted to pH 9.6 at 37° C. using 2 M sodium hydroxide (see Section 10.7.1.1). The volume was adjusted to 145 mL with 0.025 M glycine pH 9.6 solution. Working substrate was prepared freshly every day.
(309) 10.7.2 LVL-RecAP Spike Solution (500 μg/mL)
(310) A recAP spike solution were prepared by diluting 252.5 μL LVL-RecAP (Section 10.6.1) to 5.00 mL with enzyme diluent buffer (Section 10.7.1.5). The final concentration of the spike solution is 500 μg/mL, this solution was used for preparation of the recAP spiked human serum samples (Section 10.7.4).
(311) 10.7.3 RecAP Spike Solution (500 μg/mL)
(312) A RecAP spike solution was prepared by diluting 188.0 μL RecAP (Section 10.6.1) to 5.00 mL with enzyme diluent buffer (Section 10.7.1.5). The final concentration of the spike solution is 500 μg/mL, this solution was used for preparation of the RecAP spiked human serum samples (Section 10.7.5).
(313) 10.7.4 Preparation of LVL-recAP Spiked Human Serum Samples
(314) Serum samples were prepared by spiking LVL-RecAP to three individual blank serum batches using the following concentration:
(315) TABLE-US-00021 LVL-RecAP Calculated Spike Total serum Concentration activity volume volume samples (μg/mL) (U/L) (μL) (mL) 1 10.0 6603 100 5.00 2 8.00 5282 80.0 5.00 3 6.00 3962 60.0 5.00 4 4.00 2641 40.0 5.00 5 2.00 1321 20.0 5.00 6 1.00 660 10.0 5.00 7 endogenous endogenous 0 5.00 The serum samples were stored at nominal −70° C. until analysis.
10.7.5 Preparation of RecAP Spiked Human Serum Samples
(316) Serum samples were prepared by spiking RecAP to three individual blank serum batches using the following concentration:
(317) TABLE-US-00022 RecAP Calculated Spike Total serum Concentration activity volume volume samples (μg/mL) (U/L) (μL) (mL) 1 9.00 6680 90.0 5.00 2 7.20 5344 72.0 5.00 3 5.40 4088 54.0 5.00 4 3.60 2672 36.0 5.00 5 1.80 1336 18.0 5.00 6 0.900 668 9.00 5.00 7 endogenous endogenous 0 5.00 The serum samples were stored at nominal −70° C. until analysis.
10.7.6 Preparation of Sample Solutions for LVL-RecAP and RecAP Enzyme Activity
(318) The sample solutions for LVL-RecAP and RecAP enzyme activity were prepared by dilution of the LVL-RecAP product sample or RecAP product sample using enzyme diluent buffer (Section 10.7.1.5 for 25° C. method or Section 10.7.1.9 for 37° C. method).
(319) Sample 1 from LVL-RecAP and/or RecAP sample was diluted by taking 125 μL of the LVL-RecAP and/or RecAP serum sample and diluted to 2.50 mL with enzyme diluent buffer to prepare the final sample solution for LVL-RecAP or RecAP enzyme activity.
(320) Sample 2 from LVL-RecAP and/or RecAP sample was diluted by taking 125 μL of the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL with enzyme diluent buffer to prepare the final sample solution for LVL-RecAP or RecAP enzyme activity.
(321) Sample 3 from LVL-RecAP and/or RecAP sample was diluted by taking 167 μL of the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL with enzyme diluent buffer to prepare the final sample solution for LVL-RecAP or RecAP enzyme activity.
(322) Sample 4 from LVL-RecAP and/or RecAP sample was diluted by taking 250 μL of the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL with enzyme diluent buffer to prepare the final sample solution for LVL-RecAP or RecAP enzyme activity.
(323) Sample 5 from LVL-RecAP and/or RecAP sample was diluted by taking 500 μL of the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL with enzyme diluent buffer to prepare the final sample solution for LVL-RecAP and/or RecAP enzyme activity.
(324) Sample 6 from LVL-RecAP and/or RecAP sample was diluted by taking 1000 μL of the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL with enzyme diluent buffer to prepare the final sample solution for LVL-RecAP and/or RecAP enzyme activity.
(325) The endogenous (sample 7) from LVL-RecAP and/or RecAP sample was diluted by taking 1000 μL of the LVL-RecAP and/or RecAP serum sample and diluted to 2.00 mL with enzyme diluent buffer to prepare the final sample solution for LVL-RecAP and/or RecAP enzyme activity.
(326) 10.8. Execution, Results and Discussion
(327) 10.8.1 Equipment and Settings
(328) The following method and settings were used:
(329) Spectrophotometer: Thermo Fisher Evolution 300 UV/VIS with single cell Peltier heater set at 25° C. (AN-18-3) or 37° C. (AN-18-4) with magnetic stirrer
(330) Wavelength: 405 nm
(331) Measurement: For 3 minutes, each 15 seconds a measurement. The first minute was not taken into account for the calculations.
(332) Cuvette type: Glass
(333) The following solutions were pipetted into a glass cuvette. The temperature of the solutions was 25° C.±0.5° C. for AN-18-3 or 37° C.±0.5° C. for AN-18-4.
(334) TABLE-US-00023 Reagent Sample solution Blank Working substrate 1450 μL 1450 μL Enzyme diluent 50.0 μL Sample 50.0 μL Total volume 1500 μL 1500 μL
(335) First the working substrate and the enzyme diluent were mixed before the sample was added. The solution was mixed and the cuvette was placed immediately in the spectrophotometer and the increase in absorbance was measured from 1 to 3 minutes in steps of 15 seconds to obtain at least 9 data points. At the 3 minute (last) data point the Optical density was ≤1.5. Furthermore linearity was acceptable; the correlation coefficient (r) for each replicate should be ≥0.990. All sample results with a correlation coefficient (r) ≥0.990 were taken into account for the evaluation, sample results with a correlation coefficient (r)<0.990 were reported for information only. The enzyme activity was performed in duplicate mode, one test of each dilution.
(336) 10.8.2 LVL-RecAP and RecAP Enzyme Activity
(337) Although in the Study Plan was described that the difference for the two individual results (enzyme activity) should be ≤5.0% to accept the two individual dilution results, all results with a correlation coefficient (r) for each measurement ≥0.990 were used for evaluation. This because the enzyme activity method was validated for LVL-RecAP drug product samples at 25° C.±0.5° C. and not for other Alkaline Phosphatase origin and/or other conditions.
(338)
(339)