p2x1 Receptor (IC50 = 68 nM); P2X2 Receptor (IC50 = 214 nM)

Na+/Ca2+ exchanger reverse mode (NCXREV) is inhibited by PPADS tetrasodiuma (1-30 μM; 10-50 min) in a concentration- and time-dependent manner [2]. Tetrasodium PPADS is efficient against recombinant and other native P2XRs. Human P2XRs' sensitivity to PPADS tetrasodium varied depending on the subtype; the most sensitive subtypes were hP2X1, -2, -3, -5, and -7R, whereas hP2X4R had IC50s of around 1-3 and ~30 μM, respectively [3].

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In cultured mesangial cells, PPADS inhibited nucleotide, but not FCS-stimulated proliferation in a dose-dependent manner. In the anti-Thy1 model, PPADS specifically and dose-dependently reduced early (day 3), but not late (day 8), glomerular mesangial cell proliferation as well as phenotypic activation of the mesangium and slightly matrix expansion. While no consistent effect was obtained in regard to the degree of mesangiolysis, influx of inflammatory cells, proteinuria or blood pressure, PPADS treatment increased serum creatinine and urea in anti-Thy1 rats. P2Y receptor expression (P2Y2 and P2Y6) was detected in cultured MC and isolated glomeruli, and demonstrated a transient marked increase during anti-Thy1 disease. Conclusion: These data strongly suggest an in vivo role for extracellular nucleotides in mediating early MC proliferation after MC injury [4].

Mesangial cell (MC) proliferation in vivo in mesangial proliferative nephritis is inhibited by PPADS tetrasodiuma (15–60 mg/100g body weight (BW); intraperitoneal injection; every 12 hours for 8 days) without affecting non-MC in vivo proliferation [4].

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The Na(+)/Ca(2+)exchanger (NCX) principal function is taking 1 Ca(2+) out of the cytoplasm and introducing 3 Na(+). The increase of cytoplasmic Na(+) concentration induces the NCX reverse mode (NCX(REV)), favoring Ca(2+) influx. NCX(REV) can be inhibited by: KB-R7943 a non-specific compound that blocks voltage-dependent and store-operated Ca(2+) channels; SEA0400 that appears to be selective for NCX(REV), but difficult to obtain and SN-6, which efficacy has been shown only in cardiomyocytes. We found that PPADS, a P2X receptor antagonist, acts as a NCX(REV) inhibitor in guinea pig tracheal myocytes. In these cells, we characterized the NCX(REV) by substituting NaCl and NaHCO(3) with LiCl, resulting in the increase of the intracellular Ca(2+) concentration ([Ca(2+)]i) using fura 2-AM. We analyzed 5 consecutive responses of the NCX(REV) every 10 min, finding no differences among them. To evaluate the effect of different NCX(REV) blockers, concentration response curves to KB-R7943 (1, 3.2 and 10 μM), and SN-6 (3.2, 10 and 30 μM) were constructed, whereas PPADS effect was characterized as time- and concentration-dependent (1, 3.2, 10 and 30 μM). PPADS had similar potency and efficacy as KB-R7943, whereas SN-6 was the least effective. Furthermore, KCl-induced contraction, sensitive to D600 and nifedipine, was blocked by KB-R7943, but not by PPADS. KCl-induced [Ca(2+)]i increment in myocytes was also significantly decreased by KBR-7943 (10 μM). Our results demonstrate that PPADS can be used as a reliable pharmacological tool to inhibit NCX(REV), with the advantage that it is more specific than KB-R7943 because it does not affect L-type Ca(2+) channels.[1]

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Neuropathic pain consequent to peripheral injury is associated with local inflammation and overexpression of nitric oxide synthases (NOS) and inflammatory cytokines in locally recruited macrophages, Schwann and glial cells. We investigated the time course and localization of nitric oxide synthases (NOS) and cytokines in the central (spinal cord and thalamus) and peripheral nervous system (nerve and dorsal root ganglia), in a mouse model of mononeuropathy induced by sciatic nerve chronic constriction injury. ATP is recognized as an endogenous pain mediator. Therefore we also evaluated the role of purinergic signalling in pain hypersensitivity employing the P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), on pain behaviour, NOS and cytokines. The PPADS daily administration starting on day 3 after injury dose- and time-dependently decreased both tactile allodynia and thermal hyperalgesia. PPADS (25mg/kg) completely reversed nociceptive hypersensitivity and simultaneously reduced the increased NO/NOS system and IL-1beta in both peripheral (injured sciatic nerve and L4-L6 ipsilateral dorsal root ganglia) and central steps of nervous system (L4-L6 spinal cord and thalamus) involved in pain signalling. IL-6 was overexpressed only in the peripheral nervous system and PPADS prolonged administration reduced it in sciatic nerve. In conclusion, we hypothesize that NO/NOS and IL-1beta have a pronociceptive role in this neuropathy model, and that purinergic antagonism reduces pain hypersensitivity by inhibiting their overactivity[3].

Electrophysiology [2]
Nucleotide-evoked membrane currents were recorded from cRNA-injected oocytes studied under voltage-clamp conditions using a twin-electrode amplifier. Intracellular microelectrodes had a resistance of 1–2 MΩ when filled with KCl (3 M). Oocytes were perfused constantly (at 5 ml min−1) with an extracellular solution containing (mM): NaCl 110, KCl 2.5, HEPES 5, BaCl2 1.8, pH 7.4–7.5. All recordings were made at room temperature (18°C) at a holding potential between −60 and −90 mV. Electrophysiological data were filtered initially at 3 kHz, captured at a rate of 20 Hz on a computer connected to an MP100WSW interface and displayed using commercial software.

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Evaluation of IL-1β content in dorsal spinal cord by enzyme-linked immunosorbent assay (ELISA) [3]
Quantitative determination of IL-1β protein was performed, by using enzyme-linked immunosorbent assay on spinal cord from sham, CCI and PPADS-treated CCI animals. Sections of L4–L6 spinal cord were harvested as described above, flash frozen and stored at −80°C. Samples were homogenized in 0.25ml of ice-cold phosphate-buffered saline, containing protease inhibitor cocktail and centrifuged. The supernatant was used to measure IL-1β levels. Pellets were utilized for total protein determination by mean of Lowry’s method. For IL-1β measurements a CytoSet Elisa kit for mouse IL-1β was used. The concentrations of the capture and of the secondary biotinylated antibodies were 1.25 and 0.125μg/ml, respectively. Standard curves generated from recombinant protein ranged from 15 to 1000pg/ml. Streptavidin peroxidase and tetramethylbenzidine were used for colour development. The colour reaction was stopped with 2N H2SO4 and read as optical density at 450nm.

Oocyte Preparation and P2X Receptor Expression [2]
Xenopus laevis were anaesthetised with Tricaine (0.2%, wt/vol) and killed by decapitation (in accordance with Institution regulations). The dissection and removal of ovaries, as well as the preparation of defolliculated Xenopus oocytes, have been described in detail elsewhere [King et al., 1997]. Defolliculated oocytes do not possess native P1 or P2 receptors that could otherwise complicate the analysis of agonist activity [King et al., 1996a,b]. Also, defolliculated oocytes are largely devoid of ecto-ATPase activity, so avoiding the complicating issue of ectoenzyme inhibition by P2 receptor antagonists [Ziganshin et al., 1995]. Mature oocytes (stages V and VI) were injected (40 nl) cytosolically with capped ribonucleic acid (cRNA, 1 mg/ml) encoding either rat P2X1 or rat P2X3 receptor subunits. Injected oocytes were incubated at 18°C in a bathing solution (pH 7.5) containing (mM): NaCl 110, KCl 1, NaHCO3 2.4, Tris-HCl 7.5, Ca(NO3)2 0.33, CaCl2 0.41, MgSO4 0.82, supplemented with gentamycin sulphate 50 μg/l for 48 h to allow full receptor expression, then stored at 4°C for up to 12 days.

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The effects of PPADS on nucleotide- or fetal calf serum (FCS)-stimulated proliferation of cultured MC were measured by cell counting and [3H]thymidine incorporation assay. After induction of the anti-Thy1 model, rats received injections of the P2-receptor antagonist PPADS at different doses (15, 30, 60 mg/kg BW). Proliferating mesangial and non-mesangial cells, mesangial cell activation, matrix accumulation, influx of inflammatory cells, mesangiolysis, microaneurysm formation, and renal functional parameters were assessed during anti-Thy1 disease. P2Y-mRNA and protein expression was assessed using RT-PCR and real time PCR, Northern blot analysis, in situ hybridization, and immunohistochemistry[4].

Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rats, body weight 160 to 200 g (anti-Thy1 disease model) [4]
Doses: 15 mg/100g BW, 30 mg/100g BW, 60 mg/100g BW
Route of Administration: intraperitoneal (ip) injection; every 12 hrs (hrs (hours)) for 8 days (The first PPADS injection was given 60 minutes after disease induction, and the loading dose always contained double the amount of PPADS compared with subsequent injections.)
Experimental Results: Early (day 3) specifically And dose-dependently diminished mesangial cell proliferation without changing non-MC proliferation.

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Drug treatment [3]
Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid tetrasodium salt (PPADS) was dissolved in saline and used at doses of 6.25, 12.5 and 25mg/kg (0.1ml/10g). Doses were chosen according to those employed by Gourine et al. (2005) in order to attenuate fever and cytokine responses induced by lipopolysaccharide in rats. PPADS or saline was administered i.p. to neuropathic and sham-operated mice once a day for 11 days, starting from the third day after surgery. The effect of the acute administration of PPADS at the highest dose (25mg/kg) has been evaluated at both third and 14th day after lesion: behavioural evaluations were performed both 1 and 24h after administration. The same experimental protocol was applied in mice treated for 10 days with saline, i.e. 14 days after sciatic nerve ligation.

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Thermal hyperalgesia and mechanical allodynia [3]
Responses to thermal and mechanical stimuli were measured before and 3, 7 and 14 days (24h after the last administration with PPADS or saline) after the surgical procedure. Measurements were performed on both the ipsilateral and contralateral hind paws of all mice by researchers who were blind to treatments. Thermal hyperalgesia was tested according to the Hargreaves procedure (Hargreaves et al., 1988), slightly modified by us for mouse, using a Plantar test apparatus. Briefly, mice were placed in smaller clear plexiglass cubicles and allowed to acclimatize. A constant intensity radiant heat source (beam diameter 0.5cm and intensity 20 I.R.) was aimed at the midplantar area of the hind paw. The time, in seconds (s), from initial heat source activation until paw withdrawal was recorded. Mechanical allodynia was assessed using the Dynamic Plantar Aesthesiometer. Animals were placed in a test cage with a wire mesh floor, and the rigid tip of a von Frey filament (punctate stimulus) was applied to the skin of the midplantar area of the hind paw. The filament exerted an increasing force, ranging up to 5g in 20s, starting below the threshold of detection and increasing until the animal removed its paw. Withdrawal threshold was expressed in grams. Withdrawal threshold of ipsilateral and contralateral paws was measured four times and the value was the mean of the four evaluations.

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Biochemical evaluations [3]
The biochemical evaluations were performed on animals receiving the highest dose of PPADS (25mg/kg) always by researchers who were blind to treatments. At 3, 7 and 14 days following surgery, 24h after the last dose of saline or PPADS, nociceptive and mechanical thresholds were recorded. Immediately after behavioural evaluations, mice were anaesthetized with sodium pentobarbital (60mg/kg, i.p., 0.1ml/10g) and under dissecting microscope the ipsilateral sciatic nerve, proximal to the trifurcation (about 1cm), before the three ligatures in the CCI animals, the ipsilateral L4, L5 and L6 DRG, the lumbar dorsal spinal cord at L4–L6 level, and ipsilateral and contralateral thalamus were removed and immediately frozen in liquid nitrogen and stored at −80°C until the NOSs content and cytokine expression assay. In some experiments a small portion of ipsilateral sciatic nerve, proximal to the trifurcation, before three ligatures in the CCI animals, and lumbar spinal dorsal at L4–L6 level was used to prepare nuclear extracts, which were stored at −80°C until the transcription factor NF-κB was assayed. In other experiments, a small portion of sciatic nerve, between the ligatures in CCI animals and trifurcation, was stored at −80°C until the assay of myelin proteins. In view of the technical difficulty of measurement of NO, which requires accuracy in the time of sampling and prompt measurement immediately after sampling, due to the instability of NO and nitrite, we evaluated the level of NOSs (inducible and neuronal) to represent NO changes, as previously reported by Salake et al. (2000) and Wang et al. (2004).

[1]. PPADS, a P2X receptor antagonist, as a novel inhibitor of the reverse mode of the Na⁺/Ca²⁺ exchanger in guinea pig airway smooth muscle. Eur J Pharmacol. 2012 Jan 15;674(2-3):439-44.

[2]. Mapping the binding site of the P2X receptor antagonist PPADS reveals the importance of orthosteric site charge and the cysteine-rich head region. J Biol Chem. 2018 Aug 17;293(33):12820-12831.

[3]. The purinergic antagonist PPADS reduces pain related behaviours and interleukin-1 beta, interleukin-6, iNOS and nNOS overproduction in central and peripheral nervous system after peripheral neuropathy in mice. Pain. 2008 Jul;137(1):81-95.

[4]. P2 receptor antagonist PPADS inhibits mesangial cell proliferation in experimental mesangialproliferative glomerulonephritis. Kidney Int. 2002 Nov;62(5):1659-71.

[5]. PPADS is a reversible competitive antagonist of the NAADP receptor. Cell Calcium. 2007 Jun;41(6):505-11.

[6]. Actions of a Series of PPADS Analogs at P2X1 and P2X3 Receptors. Drug Dev Res. 2001 Aug;53(4):281-291.

[7]. Einfluss von ATP und seinen Derivaten auf die Aktivierung von Monozyten.

Tetrasodium 5'-phosphonatopyridoxal-6-azobenzene-2,4-disulfonate is an organic sodium salt that is the tetrasodium salt of 5'-phosphopyridoxal-6-azobenzene-2,4-disulfonic acid It has a role as a purinergic receptor P2X antagonist. It is an organic sodium salt and an organosulfonate salt. It contains a 5'-phosphonatopyridoxal-6-azobenzene-2,4-disulfonate.

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5'-phosphopyridoxal-6-azobenzene-2,4-disulfonic acid is an arenesulfonic acid that is pyridoxal 5'-phosphate carrying an additional 2,4-disulfophenylazo substituent at position 6. It has a role as a purinergic receptor P2X antagonist. It is an arenesulfonic acid, a member of azobenzenes, a member of methylpyridines, a monohydroxypyridine, a pyridinecarbaldehyde and an organic phosphate. It is functionally related to a pyridoxal 5'-phosphate. It is a conjugate acid of a 5'-phosphonatopyridoxal-6-azobenzene-2,4-disulfonate.

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Platelet Aggregation Inhibitors: Drugs or agents which antagonize or impair any mechanism leading to blood platelet aggregation, whether during the phases of activation and shape change or following the dense-granule release reaction and stimulation of the prostaglandin-thromboxane system.

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ATP is the native agonist for cell-surface ligand-gated P2X receptor (P2XR) cation channels. The seven mammalian subunits (P2X1-7) form homo- and heterotrimeric P2XRs having significant physiological and pathophysiological roles. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) is an effective antagonist at most mammalian P2XRs. Lys-249 in the extracellular domain of P2XR has previously been shown to contribute to PPADS action. To map this antagonist site, we generated human P2X1R cysteine substitutions within a circle centered at Lys-249 (with a radius of 13 Å equal to the length of PPADS). We hypothesized that cysteine substitutions of residues involved in PPADS binding would (i) reduce cysteine accessibility (measured by MTSEA-biotinylation), (ii) exhibit altered PPADS affinity, and (iii) quench the fluorescence of cysteine residues modified with MTS-TAMRA. Of the 26 residues tested, these criteria were met by only four (Lys-70, Asp-170, Lys-190, and Lys-249), defining the antagonist site, validating molecular docking results, and thereby providing the first experimentally supported model of PPADS binding. This binding site overlapped with the ATP-binding site, indicating that PPADS sterically blocks agonist access. Moreover, PPADS induced a conformational change at the cysteine-rich head (CRH) region adjacent to the orthosteric ATP-binding pocket. The importance of this movement was confirmed by demonstrating that substitution introducing positive charge present in the CRH of the hP2X1R causes PPADS sensitivity at the normally insensitive rat P2X4R. This study provides a template for developing P2XR subtype selectivity based on the differences among the mammalian subunits around the orthosteric P2XR-binding site and the CRH.[2]

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Although extracellular nucleotides have been shown to confer mitogenic effects in cultured rat mesangial cells through activation of purinergic P2 receptors (P2Y receptors), thus far the in vivo relevance of these findings is unclear. Virtually all cells and in particular the dense granules of platelets contain high levels of nucleotides that are released upon cell injury or platelet aggregation. In experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), mesangiolysis and glomerular platelet aggregation are followed by a pronounced mesangial cell (MC) proliferative response leading to glomerular hypercellularity. Therefore, we examined the role of extracellular nucleotides and their corresponding receptors in nucleotide-stimulated cultured mesangial cells and in inflammatory glomerular disease using the P2 receptor antagonist PPADS.[4]

C14H10N3O12PS2-4.4[NA+]

599.3051

598.903

C, 28.06; H, 1.68; N, 7.01; Na, 15.34; O, 32.04; P, 5.17; S, 10.70

192575-19-2

Iso-PPADS tetrasodium;207572-67-6

135484645

Orange to red solid powder

3.779

1

15

5

36

918

0

KURWUCJJNVPCHT-UHFFFAOYSA-J

InChI=1S/C14H14N3O12PS2.4Na/c1-7-13(19)9(5-18)10(6-29-30(20,21)22)14(15-7)17-16-11-3-2-8(31(23,24)25)4-12(11)32(26,27)28;;;;/h2-5,19H,6H2,1H3,(H2,20,21,22)(H,23,24,25)(H,26,27,28);;;;/q;4*+1/p-4

tetrasodium;4-[[4-formyl-5-hydroxy-6-methyl-3-(phosphonatooxymethyl)pyridin-2-yl]diazenyl]benzene-1,3-disulfonate

PPADS TETRASODIUM SALT; 192575-19-2; PPADS Tetrasodium; CHEMBL1256743; Pyridoxalphosphate-6-azophenyl-2',4'- disulfonic acid tetrasodium salt; 4-[[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid tetrasodium salt; PPADS tetrasodium salt, anhydrous; 4-[2-[4-Formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]diazenyl]-1,3-benzenedisulfonic Acid Tetrasodium Salt;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~83.43 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)