KRN 7000 |
| Catalog No.: GC18180 |
KRN 7000 (α-GalCer) is a synthetic glycolipid with antitumorial and immunostimulatory.
Products are for research use only. Not for human use. We do not sell to patients.
Cas No.:158021-47-7
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
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Purity: >96.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
Va24+ Vb11+ T cells |
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Preparation Method |
For expansion of Va24+ Vb11+ T cells, total human PBMC were cultured for 7 days in IMDM, supplemented with 50 U/ml penicillin ± streptomycin, 0.01 mM 2-mercaptoethanol (2-ME), 1.6 mM L-glutamine, 25 mM HEPES, 20% human pooled serum (HPS, CLB). |
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Reaction Conditions |
Cells were cultured with 100 ng/ml of KRN7000 for 7 days. |
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Applications |
KRN7000 caused strong activation of PB Va24+ Vb11+ T cells, the vast majority of which was capable of producing IFN-c. KRN7000 up-regulated the expression of GrB, a molecule which cleaves peptide bonds after aspartic acid residues and induces apoptosis in target cells in Va24+ Vb11+ T cells. KRN7000 could be a useful agent in the modulation of immune responses and, more specially, has the potential to act as an anti-tumor agent. |
| Animal experiment [2]: | |
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Animal models |
Specific pathogen-free (SPF) male C57BL/6J mice (weight, 12–14 g; age, 3–4 weeks) |
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Preparation Method |
Mice were housed in a controlled environment (temperature, 23 ± 3°C; humidity, 55.5 ± 10%; air renovations/h, 15; light cycle (h), 12/12) with ad libitum access to water. The mouse model of obesity-associated asthma was established by feeding the mice a normal diet with a high-fat diet administered by gavage for the entire 20 weeks. The control, asthma, and A + KRN groups were fed a normal diet. |
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Dosage form |
100 μg/kg |
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Applications |
KRN7000 intervention could alter lipid metabolism and KRN7000 reduced airway inflammation in the obese asthmatic mice to a greater extent than that of the non-obese asthmatic mice. In addition, KRN7000 significantly increased the level of serum TH1 cytokines in obese asthmatic mice. Therefore, KRN7000 stimulates a TH1 polarized secretion activity of NKT cells more significantly towards low-grade systemic inflammation in the context of obesity. The strong inhibitory effect of KRN7000 on airway inflammation in the obese asthmatic mice may be achieved by enhancing the secretion of TH1 cytokines by NKT cells rather than reducing the level of TH2 cytokines. |
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References: [1]. Van Der Vliet HJ, et al. Effects of alpha-galactosylceramide (KRN7000), interleukin-12 and interleukin-7 on phenotype and cytokine profile of human Valpha24+ Vbeta11+ T cells. Immunology. 1999 Dec;98(4):557-63. [2]. Chen Y, et al. KRN7000 Reduces Airway Inflammation via Natural Killer T Cells in Obese Asthmatic Mice. Inflammation. 2021 Oct;44(5):1982-1992. |
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KRN7000[(2S,3S.4R)-l-O-(a-D-galactopyranosyl)-2-(N-hexacosanoylamino)-l,3.4-octadecanetriol] is a highly potent synthetic alpha-galactose ceramide originally isolated from an ocean sponge that is widely used in the study of tumors and autoimmune diseases due to its ability to specifically present CD1d on the cell surface and thus activate NKT cells.[1] KRN7000 is a potent activator of antigen-presenting cells which leads to stimulation of immunoregulatory cells such as T cells, natural killer T cells (NKT), and macrophages.[3]
In vitro study indicated that KRN7000 can be recognized by NKT-cell clones and can trigger proliferation, cytokine release (IL-4 and IFN-c) and cytotoxic activity. KRN7000 decreased in the percentage of Va24+ Vb11+T cells expressing CD161, indicating that CD161 does not play a major role in the cytotoxicity mediated by KRN7000-activated Va24+ Vb11+ T cells. Moreover, KRN7000-cultured Va24+Vb11+ T cells strongly expressed CD25 (IL-2Ra), it can be expected that cytokines targeting the IL-2R will have potent effects on the expansion of Va24+ Vb11+ T cells.[2]
In vivo study demonstrated that KRN7000 prevented GVHD onset in F1 mice inoculated with haploidentical C57 splenocytes with no apparent toxicity. This treatment results in the maintenance of a healthy profile and body weight and also prevents other clinical signs of GVHD. In vivo treatment with KRN7000 resulted in stimulation of the NK1.1+T cell subset, which is implicated in the suppression of immune responses directed against autoantigens and induction of tumor immunity. KRN7000 treatment in vivo also led to increased production of IL-4. Which suggests that KRN7000 treatment in vivo might prevent the onset of GVHD through upregulation of IL-4 production.[3]
KRN 7000 is a potent synthetic α-galactosylceramide, which, in association with the antigen-presenting CD1d protein, activates NKT immune cells. As these cells have important roles in the rejection of malignant tumors and in the regulation of several autoimmune diseases, KRN 7000 has activity in many diseases, including cancer, lupus, diabetes, malaria, and tuberculosis.
KRN7000 is inherently an extremely hydrophobic molecule, therefore, almost all the methods for solubilizing this material in primarily aqueous media will contain at least some detergent. For cell culture or other in vivo models, it is recommended that the KRN7000 be initially dissolved in a 2:1 mixture of chloroform and methanol. This solution should then be aliquoted into amounts suitable for a day’s use into glass vials. After aliquoting, the chloroform/methanol solvent can be evaporated off under a stream of nitrogen or argon. We recommend turning the tube during this process to try to get the KRN7000 deposited in as thin a film as possible. The thinner the KRN7000 layer is, the easier it will be to reconstitute it. These dry aliquots can then be stored under nitrogen or argon at -20°C for several months or until needed.
On the day of the experiment, use a dried aliquot and reconstitute it using any of the options listed below:
PBS with 0.5% Tween-20. Note: It will be necessary to warm at 37°C and sonicate for 2 hours or more in a water bath sonicator in order to get the material to dissolve. Heating and sonication should be done immediately prior to every use.
5.6% sucrose with 0.75% L-histidine and 0.5% Tween-20. Heating to 80°C for several minutes will be necessary with this solvent system.
100% DMSO (anhydrous). It is critical that the DMSO be completely anhydrous. Heating to 80°C may be necessary to get the KRN7000 to completely dissolve. Please note: while this method will produce a true clear solution in DMSO, the KRN7000 will be very likely to precipitate out as soon as the DMSO is diluted into aqueous media. It is recommended that the aqueous media the DMSO stock solution will be diluted in should contain 10% serum, or BSA and that the DMSO stock solution be diluted no more than 1/100. Depending upon the make-up of the aqueous buffer, there may well still be a precipitate, however the precipitate should eventually disappear with warming and repeated vortexing or sonication in a water bath sonicator. This may take some time and will likely need to be repeated each time an aliquot is thawed for use.
Depending upon the solvent system used and the final concentration of the KRN7000, the results may be either a true clear solution or a somewhat cloudy suspension. The cloudy suspensions are not a problem, and will work fine when treating cells, just make sure they are mixed well immediately before use.
References:
[1]. Chen Y, et al. KRN7000 Reduces Airway Inflammation via Natural Killer T Cells in Obese Asthmatic Mice. Inflammation. 2021 Oct;44(5):1982-1992.
[2]. Van Der Vliet HJ, et al. Effects of alpha-galactosylceramide (KRN7000), interleukin-12 and interleukin-7 on phenotype and cytokine profile of human Valpha24+ Vbeta11+ T cells. Immunology. 1999 Dec;98(4):557-63.
[3]. Morecki S, et al. Effect of KRN7000 on induced graft-vs-host disease. Exp Hematol. 2004 Jul;32(7):630-7.
| Cas No. | 158021-47-7 | SDF | |
| Chemical Name | N-[(1S,2S,3R)-1-[(α-D-galactopyranosyloxy)methyl]-2,3-dihydroxyheptadecyl]-hexacosanamide | ||
| Canonical SMILES | O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@H](NC(CCCCCCCCCCCCCCCCCCCCCCCCC)=O)[C@H](O)[C@H](O)CCCCCCCCCCCCCC | ||
| Formula | C50H99NO9 | M.Wt | 858.3 |
| Solubility | 1mg/ml in DMSO | Storage | Store at -20°C |
| General tips | For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months. | ||
| Shipping Condition | Evaluation sample solution : ship with blue ice All other available size: ship with RT , or blue ice upon request |
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Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
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Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)
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Working concentration: mg/ml;
Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid 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 master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL saline, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
The stimulating adventure of KRN 7000
Associated with the CD1d protein, KRN 7000, a potent synthetic α-galactosylceramide, is known to activate the invariant NKT immune cells. This stimulation then leads to the production of different cytokines modulating a T(H)1/T(H)2 immune response balance involved in protection against several pathologies such as autoimmune diseases and cancers. Various efforts have been made toward the synthesis of simple and more functionalized analogues in order to selectively induce T(H)1 or T(H)2-type cytokine production. Since the discovery of KRN 7000, structure-activity relationships, crystallographic and modelling studies have pointed to the potential of several GalCer analogues in term of selective bioactivity, and have highlighted interesting elements in order to better understand the recognition and activation mechanisms of immune iNKT cells. By presenting an up-to-date library of analogues, collecting recent breakthroughs done in crystallography and molecular modelling, and relating them to the available biological results, we hope that this review will highlight and help the scientific community in their KRN research.
Synthesis and biological activities of C-glycosides of KRN 7000 with novel ceramide residues
The identification of immunoactive agents for clinical and mechanistic applications is a very active area of research. In this vein, analogues of the potent immunostimulant KRN 7000 with diverse cytokine profiles have attracted considerable attention. These compounds have been shown to activate iNKT cells via presentation by CD1d. Herein, we report on the synthesis and activity for four new C-glycosides of KRN 7000, 11-phenylundecanoyl and 11-p-fluorophenylundecanoyl derivatives of C-KRN 7000, 2,3-bis-epi-C-KRN 7000 and the reverse amide of C-KRN 7000. In mice, compared to C-KRN 7000, 2,3-bis-epi-C-KRN 7000 stimulated higher release of the anti-inflammatory cytokine IL-4 and lower release of the inflammatory cytokines IFN-γ and IL-12. The phenyl terminated alkanoyl and reverse amide analogues were inactive. These data suggest that structure activity effects for KRN 7000 are not necessarily additive and their use in the design of new analogues will require an improved understanding of how subtle structural changes impact on cytokine activity.
Fully synthetic Tn-based three-component cancer vaccine using covalently linked TLR4 ligand MPLA and iNKT cell agonist KRN-7000 as built-in adjuvant effectively protects mice from tumor development
We present a new strategy for self-adjuvanting vaccine development that has different types of covalently-linked immunostimulants as the carrier molecule. Using Tn antigen as the model, a three-component vaccine (MPLA-Tn-KRN7000) containing the TLR4 ligand MPLA and the iNKT cell agonist KRN7000 was designed and synthesized. This expands fully synthetic self-adjuvanting vaccine studies that use a single carrier to one with two different types of carriers. The corresponding two-component conjugate vaccines Tn-MPLA, Tn-KRN7000 and Tn-CRM197 were also synthesized, as controls. The immunological evaluation found that MPLA-Tn-KRN7000 elicits robust Tn-specific and T cell-dependent immunity. The antibodies specifically recognized, bound to and exhibited complement-dependent cytotoxicity against Tn-positive cancer cells. In addition, MPLA-Tn-KRN7000 increased the survival rate and survival time of tumor-challenged mice, and surviving mice reject further tumor attacks without any additional treatment. Compared to the glycoprotein vaccine Tn-CRM197, the two-component conjugate vaccines, Tn-MPLA and Tn-KRN7000, and the physical mixture of Tn-MPLA and Tn-KRN7000, MPLA-Tn-KRN7000 showed the most effect at combating tumor cells both in vitro and in vivo. The comparison of immunological studies in wild-type and TLR4 knockout mice, along with the test of binding affinity to CD1d protein suggests that the covalently linked MPLA-KRN7000 immunostimulant induces a synergistic activation of TLR4 and iNKT cell that improves the immunogenicity of Tn. This work demonstrates that MPLA-Tn-KRN7000 has the potential to be a vaccine candidate and provides a new direction for fully synthetic vaccine design.
Use of the NEO strategy (Nucleophilic addition/Epoxide Opening) for the synthesis of a new C-galactoside ester analogue of KRN 7000
Our goal in the search for potentially bioactive analogues of KRN 7000 was to design an easy synthetic approach to a library of analogues using a strategy recently developed in our laboratory based on a Nucleophilic addition followed by an Epoxide Opening (the NEO strategy). Through the use of a common pivotal structure, a new C-galactoside ester analogue (23) was synthesized which showed an encouraging T(H)2 biased response during preliminary biological tests.
KRN7000 Reduces Airway Inflammation via Natural Killer T Cells in Obese Asthmatic Mice
Although natural killer T cells (NKT cells) are altered in obese asthmatic mice, their function remains completely unclear. To further explore the potential mechanism of NKT cells in airway inflammation of obesity-associated asthma, we examined the effects of α-galactosylceramide (KRN7000) on airway inflammation in obese asthmatic mice. Male C57BL/6J mice were divided into five groups: (1) control; (2) asthma; (3) A + KRN, asthma with KRN7000; (4) obese asthma; and (5) OA + KRN, obese asthma with KRN7000. Cytometric bead array (CBA) was used to detect interleukin-4 (IL-4), IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the serum. Flow cytometry was used to detect NKT cells and CD69+ NKT cells. Airway inflammation was observed in pathological sections, and calmodulin (CaM) expression was observed by immunohistochemistry in lung tissues. Airway inflammation in the obese asthma group was more severe than that of the asthma group. Airway inflammation of the OA + KRN group was reduced more than that of the A + KRN group. CD69+ NKT cells were only significantly reduced in the OA + KRN group. The levels of serum IFN-γ and TNF-α increased more in the OA + KRN group than in the A + KRN group. CaM is widely expressed in the cytoplasm of the lung tissues and was sharply decreased in the OA + KRN group. KRN7000 can significantly reduce airway inflammation in obesity-associated asthma by regulating NKT cell cytokine secretion and intracellular calcium. These results may contribute to the development of novel therapeutic approaches.
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