Size | Price | Stock | Qty |
---|---|---|---|
5mg |
|
||
10mg |
|
||
25mg |
|
||
50mg |
|
||
100mg |
|
||
Other Sizes |
|
Purity: ≥98%
CL2A-SN-38, an SN-38 (topoisomerase-I inhibitor) analogue, is a peptide-based drug linker used to synthesize antibody drug conjugates (ADC). It is composed of a potent DNA Topoisomerase I inhibitor SN-38 and a linker CL2A, which provides significant and specific anticancer effects against a panel of human solid tumors. CL2A-SN-38 is conjugated to the anti-Trop-2-humanized antibody, SN-38-anti-Trop-2 antibody-drug conjugate (ADC) against several human solid tumor types, provides significant and specific antitumor effects against a range of human solid tumor types.
Targets |
Camptothecins/DNA Topoisomerase I
|
---|---|
ln Vitro |
Two SN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to the anti-Trop-2-humanized antibody, hRS7. The immunoconjugates were characterized in vitro for stability, binding, and cytotoxicity [1].
In vitro cytotoxicity studies demonstrated that hRS7-CL2A-SN-38 had IC50 values in the nM range against several different solid tumor lines (Table 1). The IC50 with free SN-38 was lower than the conjugate in all cell lines. While there was no correlation between Trop-2 expression and sensitivity to hRS7-CL2A-SN-38, the IC50 ratio of the ADC vs. free SN-38 was lower in the higher Trop-2-expressing cells, most likely reflecting the enhanced ability to internalize the drug when more antigen is present. SN-38 is known to activate several signaling pathways in cells, leading to apoptosis (34–37). Our initial studies examined the expression of two proteins involved in early signaling events (p21Waf1/Cip1 and p53) and one late apoptotic event (cleavage of poly-ADP-ribose polymerase (PARP)) in vitro (Fig. 2). In BxPC-3 (Fig. 2A), SN-38 led to a 20-fold increase in p21Waf1/Cip1 expression, while hRS7-CL2A-SN-38 resulted in only a 10-fold increase, a finding consistent with the higher activity with free SN-38 in this cell line (Table 1). However, hRS7-CL2A-SN-38 increased p21Waf1/Cip1 expression in Calu-3 more than 2-fold over free SN-38 (Fig. 2B). A greater disparity between hRS7-CL2A-SN-38- and free SN-38-mediated signaling events was observed in p53 expression. In both BxPC-3 and Calu-3, up-regulation of p53 with free SN-38 was not evident until 48 h, while hRS7-CL2A-SN-38 up-regulated p53 within 24 h. Additionally, p53 expression in cells exposed to the ADC was higher in both cell lines compared to SN-38. Interestingly, while hRS7 IgG had no appreciable effect on p21Waf1/Cip1 expression, it did induce the up-regulation of p53 in both BxPC-3 and Calu-3, but only after a 48-h exposure. In terms of later apoptotic events, cleavage of PARP was evident in both cell lines when incubated with either SN-38 or the conjugate (Fig. 2C). The presence of the cleaved PARP was higher at 24 h in BxPC-3, which correlates with high expression of p21 and its lower IC50. The higher degree of cleavage with free SN-38 over the ADC was consistent with the cytotoxicity findings [1]. |
ln Vivo |
In mice bearing human colon (COLO 205) or pancreatic (Capan-1) tumor xenografts, CL2A-SN-38 in combination with the anti-Trop-2 humanized antibody hRS7 (intraperitoneal injection, 0.2 or 0.4 mg/kg, twice weekly, 4 weeks) can both significantly inhibit tumor growth[1].
Efficacy of hRS7-SN-38 [1] Since Trop-2 is widely expressed in several human carcinomas, studies were performed in several different human cancer models, which started with an evaluation of the hRS7-CL2-SN-38 linkage, but later, conjugates with the CL2A-linkage were used. Calu-3-bearing nude mice given 0.04 mg SN-38/kg of the hRS7-CL2-SN-38 every 4 days x 4 had a significantly improved response compared to animals administered the equivalent amount of hLL2-CL2-SN-38 (TV=0.14 ± 0.22 cm3 vs. 0.80 ± 0.91 cm3, respectively; AUC42days P<0.026) (Fig. 3A). A dose-response was observed when the dose was increased to 0.4 mg/kg SN-38. At this higher dose level, all mice given the specific hRS7 conjugate were ‘cured’ within 28 days, and remained tumor-free until the end of the study on day 147, while tumors re-grew in animals treated with the irrelevant ADC (specific vs. irrelevant AUC98days: P=0.05). In mice receiving the mixture of hRS7 IgG and SN-38, tumors progressed >4.5-fold by day 56 (TV=1.10 ± 0.88 cm3; AUC56days P<0.006 versus hRS7-CL2-SN-38). Efficacy also was examined in human colonic (COLO 205) and pancreatic (Capan-1) tumor xenografts. In COLO 205 tumor-bearing animals, (Fig. 3B), hRS7-CL2-SN-38 (0.4 mg/kg, q4dx8) prevented tumor growth over the 28-day treatment period with significantly smaller tumors compared to control anti-CD20 ADC (hA20-CL2-SN-38), or hRS7 IgG (TV=0.16 ± 0.09 cm3, 1.19 ± 0.59 cm3, and 1.77 ± 0.93 cm3, respectively; AUC28days P<0.016). The MTD of irinotecan (24 mg SN-38/kg, q2dx5) was as effective as hRS7-CL2-SN-38, since mouse serum can more efficiently convert irinotecan to SN-38 (38–41) than human serum, but the SN-38 dose in irinotecan (2400 μg cumulative) was 37.5-fold greater than with the conjugate (64 μg total). Animals bearing Capan-1 showed no significant response to irinotecan alone when given at an SN-38-dose equivalent to the hRS7-CL2-SN-38 conjugate (e.g., on day 35, average tumor size was 0.04 ± 0.05 cm3 in animals given 0.4 mg SN-38/kg hRS7-SN-38 vs. 1.78 ± 0.62 cm3 in irinotecan-treated animals given 0.4 mg/kg SN-38; AUCday35 P<0.001) (Fig. 3C). When the irinotecan dose was increased 10-fold to 4 mg/kg SN-38, the response improved, but still was not as significant as the conjugate at the 0.4 mg/kg SN-38 dose level (TV=0.17 ± 0.18 cm3 vs. 1.69 ± 0.47 cm3, AUCday49 P<0.001). An equal dose of non-targeting hA20-CL2-SN-38 also had a significant anti-tumor effect as compared to irinotecan-treated animals, but the specific hRS7 conjugate was significantly better than the irrelevant ADC (TV=0.17 ± 0.18 cm3 vs. 0.80 ± 0.68 cm3, AUCday49 P<0.018). Studies with the hRS7-CL2A-SN-38 ADC were then extended to two other models of human epithelial cancers. In mice bearing BxPC-3 human pancreatic tumors (Fig. 3D), hRS7-CL2A-SN-38 again significantly inhibited tumor growth in comparison to control mice treated with saline or an equivalent amount of non-targeting hA20-CL2A-SN-38 (TV=0.24 ± 0.11 cm3 vs. 1.17 ± 0.45 cm3 and 1.05 ± 0.73 cm3, respectively; AUCday21 P<0.001), or irinotecan given at a 10-fold higher SN-38 equivalent dose (TV=0.27 ± 0.18 cm3 vs. 0.90 ± 0.62 cm3, respectively; AUCday25 P<0.004). Interestingly, in mice bearing SK-MES-1 human squamous cell lung tumors treated with 0.4 mg/kg of the ADC (Fig. 3E), tumor growth inhibition was superior to saline or unconjugated hRS7 IgG (TV=0.36 ± 0.25 cm3 vs. 1.02 ± 0.70 cm3 and 1.30 ± 1.08 cm3, respectively; AUC28 days, P<0.043), but non-targeting hA20-CL2A-SN-38 or the MTD of irinotecan provided the same anti-tumor effects as the specific hRS7-SN-38 conjugate. |
Enzyme Assay |
Preparations of CL2A-SN-38 (M.W. 1480) and its hRS7 conjugate, and stability, binding and cytotoxicity studies, were conducted as described previously, and are presented in the Supplemental Data. Cell lysates were prepared and immunoblotting for p21Waf1/Cip, p53, and PARP (poly-ADP-ribose polymerase) was done as described in Supplemental Data. Concentrations, timing, and primary antibodies are shown in the figure legends [1].
|
Cell Assay |
All human cancer cell lines used in this study were purchased from the American Type Culture Collection. These include Calu-3 (non-small cell lung carcinoma), SK-MES-1 (squamous cell lung carcinoma), COLO 205 (colonic adenocarcinoma), Capan-1 and BxPC-3 (pancreatic adenocarcinomas), and PC-3 (prostatic adenocarcinomas). Humanized RS7 IgG and control humanized anti-CD20 (hA20 IgG, veltuzumab) and anti-CD22 (hLL2 IgG, epratuzumab) antibodies were prepared at Immunomedics, Inc. Irinotecan (20 mg/mL) was obtained from Hospira, Inc.[1].
|
Animal Protocol |
For all animal studies, the doses of SN-38 immunoconjugates and irinotecan are shown in SN-38 equivalents. Based on a mean SN-38/IgG substitution ratio of six, a dose of 500 μg ADC to a 20-gram mouse (25 mg/kg) contains 0.4 mg/kg of SN-38. Irinotecan doses are likewise shown as SN-38 equivalents (i.e., 40 mg irinotecan/kg is equivalent to 24 mg/kg of SN-38).
NCr female athymic nude (nu/nu) mice, 4–8 weeks old, and male Swiss-Webster mice, 10 weeks old, were purchased from Taconic Farms (Germantown, NY). All animal studies were approved by the Center for Molecular Medicine and Immunology’s Institutional Animal Care and Use Committee (IACUC). Tolerability studies were performed in Cynomolgus monkeys (Macaca fascicularis; 2.5–4 kg male and female) by SNBL USA, Ltd. after approval by SNBL USA’s IACUC. Animals were implanted subcutaneously with different human cancer cell lines as described in the Supplemental Information. Tumor volume (TV) was determined by measurements in two dimensions using calipers, with volumes defined as: L x w2/2, where L is the longest dimension of the tumor and w the shortest. Tumors ranged in size between 0.10 to 0.47 cm3 when therapy began. Treatment regimens, dosages, and number of animals in each experiment are described in the Results. The lyophilized hRS7-CL2A-SN-38 and control ADC were reconstituted and diluted as required in sterile saline. All reagents were administered intraperitoneally (0.1 mL), except irinotecan, which was administered intravenously. The dosing regimen was influenced by our prior investigations, where the ADC was given every 4 days or twice weekly for varying lengths of time. This dosing frequency reflected a consideration of the conjugate’s serum half-life in vitro, in order to allow a more continuous exposure to the ADC. |
ADME/Pharmacokinetics |
Biodistribution of hRS7-CL2A-SN-38 [1]
The biodistributions of hRS7-CL2A-SN-38 or unconjugated hRS7 IgG were compared in mice bearing SK-MES-1 human squamous cell lung carcinoma xenografts (Suppl. Table S1), using the respective 111In-labeled substrates. A pharmacokinetic analysis was performed to determine the clearance of hRS7-CL2A-SN-38 relative to unconjugated hRS7 (Fig. 4A). The ADC cleared faster than the equivalent amount of unconjugated hRS7, with the ADC exhibiting ~40% shorter half-life and mean residence time. Nonetheless, this had a minimal impact on tumor uptake (Fig. 4B). While there were significant differences at the 24- and 48-h time-points, by 72 h (peak uptake) the amounts of both agents in the tumor were similar. Among the normal tissues, hepatic (Fig. 4C) and splenic (Fig 4D) differences were the most striking. At 24 h post-injection, there was >2-fold more hRS7-CL2A-SN-38 in the liver than hRS7 IgG. Conversely, in the spleen there was three-fold more parental hRS7 IgG present at peak uptake (48-h time-point) than hRS7-CL2A-SN-38. Uptake and clearance in the rest of the tissues generally reflected differences in the blood concentration. Since twice-weekly doses were given for therapy, tumor uptake in a group of animals that first received a pre-dose of 0.2 mg/kg (250 μg protein) of the hRS7 ADC 3 days before the injection of the 111In-labeled antibody was examined. Tumor uptake of 111In-hRS7-CL2A-SN-38 in pre-dosed mice was substantially reduced at every time-point in comparison to animals that did not receive the pre-dose (e.g., at 72 h, pre-dosed tumor uptake was 12.5 ± 3.8% ID/g vs. 25.4 ± 8.1% ID/g in animals not given the pre-dose; P = 0.0123; Fig. 4E). Pre-dosing had no appreciable impact on blood clearance or tissue uptake (Suppl. Table S2). These studies suggest that in some tumor models, tumor accretion of the specific antibody can be reduced by the preceding dose(s), which likely explains why the specificity of a therapeutic response could be diminished with increasing ADC doses and why further dose escalation is not indicated. |
Toxicity/Toxicokinetics |
Tolerability of hRS7-CL2A-SN-38 in Swiss-Webster mice and Cynomolgus monkeys [1]
Swiss-Webster mice tolerated two doses over three days, each of 4, 8, and 12 mg SN-38/kg of the hRS7-CL2A-SN-38, with minimal transient weight loss (Suppl. Figure S2). No hematopoietic toxicity occurred and serum chemistries only revealed elevated aspartate transaminase (AST) and alanine transaminase (ALT) (Figure 5). Seven days after treatment, AST rose above normal levels (>298 U/L) in all three treatment groups (Fig. 5A), with the largest proportion of mice being in the 2 × 8 mg/kg group. However, by 15 days post-treatment, most animals were within the normal range. ALT levels were also above the normal range (>77 U/L) within seven days of treatment (Fig. 5B) and with evidence of normalization by Day 15. Livers from all these mice did not show histologic evidence of tissue damage (not shown). In terms of renal function, only glucose and chloride levels were somewhat elevated in the treated groups. At 2 × 8 mg/kg, 5 of 7 mice had slightly elevated glucose levels (range of 273 to 320 mg/dL, upper end of normal 263 mg/dL) that returned to normal by 15 days post-injection. Likewise, chloride levels were slightly elevated, ranging from 116 to 127 mmol/L (upper end of normal range 115 mmol/L) in the two highest dosage groups (57% in the 2 × 8 mg/kg group and 100% of the mice in the 2 × 12 mg/kg group), and remained elevated out to 15 days post-injection. This also could be indicative of gastrointestinal toxicity, since most chloride is obtained through absorption by the gut; however, at termination, there was no histologic evidence of tissue damage in any organ system examined (not shown). Since mice do not express Trop-2, a more suitable model was required to determine the potential of the hRS7 conjugate for clinical use. Immunohistology studies revealed binding in multiple tissues in both humans and Cynomolgus monkeys (breast, eye, gastrointestinal tract, kidney, lung, ovary, fallopian tube, pancreas, parathyroid, prostate, salivary gland, skin, thymus, thyroid, tonsil, ureter, urinary bladder, and uterus) (not shown). Based on this cross-reactivity, a tolerability study was performed in monkeys. The group receiving 2 × 0.96 mg SN-38/kg of hRS7-CL2A-SN-38 had no significant clinical events following the infusion and through the termination of the study. Weight loss did not exceed 7.3% and returned to acclimation weights by day 15. Transient decreases were noted in most of the blood count data (neutrophil and platelet data shown in Fig. 5C and 5D), but values did not fall below normal ranges. No abnormal values were found in the serum chemistries. Histopathology of the animals necropsied on day 11 (eight days after last injection) showed microscopic changes in hematopoietic organs (thymus, mandibular and mesenteric lymph nodes, spleen, and bone marrow), gastrointestinal organs (stomach, duodenum, jejunum, ileum, cecum, colon, and rectum), female reproductive organs (ovary, uterus, and vagina), and at the injection site. These changes ranged from minimal to moderate and were fully reversed at the end of the recovery period (day 32) in all tissues, except in the thymus and gastrointestinal tract, which were trending towards full recovery at this later time-point. At the 2 × 1.92 mg SN-38/kg dose level of the conjugate, there was one death arising from gastrointestinal complications and bone marrow suppression, and other animals within this group showed similar, but more severe adverse events than the 2 × 0.96 mg/kg group. These data indicate that dose-limiting toxicities were identical to that of irinotecan; namely, intestinal and hematologic. Thus, the MTD for hRS7-CL2A-SN-38 lies between 2 × 0.96 to 1.92 mg SN-38/kg, which represents a human equivalent dose of 2 × 0.3 to 0.6 mg/kg SN-38. |
References | |
Additional Infomation |
Purpose: Evaluate the efficacy of an SN-38-anti-Trop-2 antibody-drug conjugate (ADC) against several human solid tumor types, and to assess its tolerability in mice and monkeys, the latter with tissue cross-reactivity to hRS7 similar to humans.
Experimental design: Two SN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to the anti-Trop-2-humanized antibody, hRS7. The immunoconjugates were characterized in vitro for stability, binding, and cytotoxicity. Efficacy was tested in five different human solid tumor-xenograft models that expressed Trop-2 antigen. Toxicity was assessed in mice and in Cynomolgus monkeys. Results: The hRS7 conjugates of the two SN-38 derivatives were equivalent in drug substitution (∼ 6), cell binding (K(d) ∼ 1.2 nmol/L), cytotoxicity (IC(50) ∼ 2.2 nmol/L), and serum stability in vitro (t/(½) ∼ 20 hours). Exposure of cells to the ADC demonstrated signaling pathways leading to PARP cleavage, but differences versus free SN-38 in p53 and p21 upregulation were noted. Significant antitumor effects were produced by hRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P ≤ 0.05), Capan-1 (P < 0.018), BxPC-3 (P < 0.005), and COLO 205 tumors (P < 0.033) when compared to nontargeting control ADCs. Mice tolerated a dose of 2 × 12 mg/kg (SN-38 equivalents) with only short-lived elevations in ALT and AST liver enzyme levels. Cynomolgus monkeys infused with 2 × 0.96 mg/kg exhibited only transient decreases in blood counts, although, importantly, the values did not fall below normal ranges. Conclusions: The anti-Trop-2 hRS7-CL2A-SN-38 ADC provides significant and specific antitumor effects against a range of human solid tumor types. It is well tolerated in monkeys, with tissue Trop-2 expression similar to humans, at clinically relevant doses, and warrants clinical investigation.[1] |
Molecular Formula |
C77H101CL4N11O26
|
---|---|
Molecular Weight |
1738.4960
|
Exact Mass |
1479.68
|
Elemental Analysis |
C, 53.20; H, 5.86; Cl, 8.16; N, 8.86; O, 23.93
|
CAS # |
1279680-68-0
|
Related CAS # |
1279680-68-0
|
PubChem CID |
89983570
|
Appearance |
Light yellow to yellow solid powder
|
LogP |
-0.1
|
Hydrogen Bond Donor Count |
6
|
Hydrogen Bond Acceptor Count |
26
|
Rotatable Bond Count |
50
|
Heavy Atom Count |
106
|
Complexity |
2930
|
Defined Atom Stereocenter Count |
2
|
SMILES |
CCC1=C2CN3C(=CC4=C(C3=O)COC(=O)[C@@]4(CC)OC(=O)OCC5=CC=C(C=C5)NC(=O)[C@H](CCCCN)NC(=O)COCC(=O)NCCOCCOCCOCCOCCOCCOCCOCCOCCN6C=C(N=N6)CNC(=O)C7CCC(CC7)CN8C(=O)C=CC8=O)C2=NC9=C1C=C(C=C9)O
|
InChi Key |
WWSNNYDLXHTRLZ-JGQYWRMXSA-N
|
InChi Code |
InChI=1S/C73H97N11O22.2C2H2Cl2O2/c1-3-55-56-39-54(85)16-17-60(56)79-67-57(55)44-83-62(67)40-59-58(70(83)92)46-104-71(93)73(59,4-2)106-72(94)105-45-50-10-14-52(15-11-50)77-69(91)61(7-5-6-20-74)78-64(87)48-103-47-63(86)75-21-23-95-25-27-97-29-31-99-33-35-101-37-38-102-36-34-100-32-30-98-28-26-96-24-22-82-43-53(80-81-82)41-76-68(90)51-12-8-49(9-13-51)42-84-65(88)18-19-66(84)89;2*3-1(4)2(5)6/h10-11,14-19,39-40,43,49,51,61,85H,3-9,12-13,20-38,41-42,44-48,74H2,1-2H3,(H,75,86)(H,76,90)(H,77,91)(H,78,87);2*1H,(H,5,6)/t49?,51?,61-,73-;;/m0../s1
|
Chemical Name |
4-((S)-2-(4-aminobutyl)-35-(4-((4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontanamido)benzyl ((S)-4,11-diethyl-9-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) carbonate bis(2,2-dichloroacetate)
|
Synonyms |
CL2A-SN-38; CL2A-SN 38; CL2A-SN38; CL2ASN-38; CL2A SN 38; CL2ASN38; CL2A-SN38 DCA salt; CL2A-SN38 dichloroacetic acid.
|
HS Tariff Code |
2934.99.9001
|
Storage |
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 (e.g. under nitrogen), away from moisture and light. |
Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
Solubility (In Vitro) |
DMSO : ~100 mg/mL (~67.54 mM)
|
---|---|
Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (1.40 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (1.40 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (1.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10% DMSO+ 40% PEG300+ 5% Tween-80+ 45% saline: 2.08 mg/mL (1.40 mM) |
Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
1 mM | 0.5752 mL | 2.8760 mL | 5.7521 mL | |
5 mM | 0.1150 mL | 0.5752 mL | 1.1504 mL | |
10 mM | 0.0575 mL | 0.2876 mL | 0.5752 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration: mg/mL;
Method for preparing DMSO stock solution: mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation::Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O,mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.