Leishmania;Plasmodium

The infusion-related toxicity of amphotericin B, which includes fever and chills, limits its administration. This effect is thought to be caused by innate immune cells producing proinflammatory cytokines. TLR2 and CD14-expressing cells release inflammatory cytokines and undergo signal transduction when exposed to amphotericin B[1]. Amphotericin B's relative toxicity limits its usefulness as it interacts with cholesterol, the primary sterol found in mammalian membranes. In the subphase, amphotericin B is distributed either as a highly aggregated state or as a pre-micellar state[2].Amphotericin B only kills Leishmania promastigotes (LPs) that are unicellular when they form aqueous pores that are permeable to small cations and anions. A polarization potential is induced by amphotericin B (0.1 mM) in liposomes loaded with KCl and suspended in an iso-osmotic sucrose solution, signifying K+ leakage. The negative membrane potential nearly completely collapses when amphotericin B (0.05 mM) is added, indicating Na+ entry into the cells[3].

In the hamster scrapie model, amphotericin B causes the incubation period to be extended and the accumulation of PrPSc to be reduced. In mice suffering from transmissible subacute spongiform encephalopathies (TSSE), amphotericin B significantly lowers PrPSc levels[4]. In mouse malaria, amphotericin B directly affects Plasmodium falciparum and has an impact on parasitemia, host survival, and eryptosis of infected erythrocytes. In mice infected with Plasmodium berghei, amphotericin B tends to postpone the development of parasitemia and considerably postpones host death[5].

Polyfect reagent and DEAE-dextran are used to transiently transfect THP-1 and HEK293 cells, respectively. Genes encoding the NF-κB-dependent pELAM-luc luciferase reporter, TLR2, TLR4, CD14, and MD2 are present in transfected plasmids. In 12-well plates, cells (5×105 THP-1 or 1×105 HEK293) are added, and after 18 hours, they are washed and stimulated for 5 hours. Following the instructions, cells are lysed in reporter lysis buffer, and the lysates are subjected to luminescence analysis using a Monolight 3010 luminometer and Promega luciferase substrate.

AmB-induced cell death kinetics against Leishmania promastigotes are monitored via fluorometry employing ethidium bromide (EB), a compound that binds DNA. A SPEX Fluorolog II spectrophotometer is used to measure fluorescence at excitation-emission wavelengths of 365–580 nm. Promastigotes are added to a fluorescence cuvette containing 2 mL of various buffered solutions, always containing 10 mM glucose and 50 mM EB, and incubated for 5 minutes with gentle stirring at a final concentration of 25×106 cells/mL.Following the attainment of signal stabilization, AmB is introduced and dissolved in dimethylsulfoxide. Digitonin (50 mg/mL) is always added to achieve maximum EB incorporation. A buffer of 75 mM TRIS (pH 4 7.6) is applied to all solutions, which also contain 150 mM KCl (BK+), 150 mM NaCl (BNa+), 150 mM choline chloride, 100 mM sucrose, and 100 mM NaCl. A sophisticated instrument called the SW2 osmometer is always used to adjust the osmolarity of all solutions to 390±5 mOsm.

Efficacy of PEO-b-p(HASA)/AmB. Efficacy was assessed by organism killing in the kidneys of a neutropenic murine model of disseminated fungal infection as described previously by Andes et al. A clinical isolate of Candida albicans (K-1) was grown and quantified on SDA. For 24 h prior to infection, the organism was subcultured at 35 °C on SDA slants. A 106 CFU/mL inoculum (CFU, colony forming units) was prepared by placing six fungal colonies into 5 mL of sterile, depyrogenated normal (0.9%) saline warmed to 35 °C. Six-week-old ICR/Swiss specific-pathogen-free female mice were obtained from Harlan Sprague Dawley . All animal studies were approved by the Animal Research Committee of the William S. Middleton Memorial VA Hospital (Madison, WI). The mice were weighed (23−27 g) and given intraperitoneal injections of cyclophosphamide to render neutropenia. (For the purposes of this study, neutropenia was defined as <100 polymorphonuclear leukocytes/mm3.) Each mouse was dosed with 150 mg/kg of cyclophosphamide 4 days prior to infection and 100 mg/kg 1 day before infection. Disseminated candidiasis was induced via tail vein injection of 100 μL of inoculum. [5]
The AmB/polymeric micelle formulations or micelle blanks were reconstituted with 1.0 mL of 5% dextrose. The treatment group was given single 200 μL intravenous (iv) injections of reconstituted AmB/PEO-b-p(HASA), 91% 2 h postinfection. Doses were calculated in terms of mg of AmB/kg of body weight. Control animals were given a placebo of “blank” polymeric micelles. Over time, two animals per experimental condition were sacrificed by CO2 asphyxiation. The kidneys from each animal were removed and homogenized. The homogenate was diluted 10-fold with 9% saline and plated on SDA. The plates were then incubated for 24 h at 35 °C and inspected for CFU determination. The lower limit of detection for this technique is 100 CFU/mL. To compare the antifungal activity of the AmB/ micelle formulations with that of Fungizone, animals were dosed with equivalent doses of AmB as Fungizone as described above. The control animals for the Fungizone group received 200 μL iv injections of 5% dextrose. All results are expressed as the mean CFU per kidney for two animals (four kidneys total). The change in the area under the time−kill curves was calculated by ΔAUCTK = AUCcontrol − AUCtreatment. Outcomes were compared using ANOVA on ranks.[5]

[1]. The antifungal drug amphotericin B promotes inflammatory cytokine release by a Toll-like receptor- and CD14-dependent mechanism. J Biol Chem. 2003 Sep 26;278(39):37561-8. Epub 2003 Jul 14.

[2]. The effect of aggregation state of amphotericin-B on its interactions with cholesterol- or ergosterol-containing phosphatidylcholine monolayers. Chem Phys Lipids. 1997 Feb 28;85(2):145-55.

[3]. Amphotericin B kills unicellular leishmanias by forming aqueous pores permeable to small cations and anions. J Membr Biol. 1996 Jul;152(1):65-75.

[4]. Pharmacological studies of a new derivative of amphotericin B, MS-8209, in mouse and hamster scrapie. J Gen Virol. 1994 Sep;75 (Pt 9):2499-503.

[5]. Amphotericin B encapsulated in micelles based on poly(ethylene oxide)-block-poly(L-amino acid) derivatives exerts reduced in vitro hemolysis but maintains potent in vivo antifungal activity. Biomacromolecules. 2003 May-Jun;4(3):750-7.

C47H73NO17

924.09

923.4878

C, 61.09; H, 7.96; N, 1.52; O, 29.43

1397-89-3

Amphotericin B trihydrate;1202017-46-6;Amphotericin B-13C6

Light yellow to yellow solid

0

320Ų

O=C([C@@H]([C@](O1)([H])C[C@@H](O[C@@H]2O[C@H](C)[C@@H](O)[C@H](N)[C@@H]2O)/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)O3)[C@@H](O)C[C@@]1(O)C[C@@H](O)C[C@@H](O)[C@H](O)CC[C@@H](O)C[C@@H](O)CC3=O)O

APKFDSVGJQXUKY-INPOYWNPSA-N

InChI=1S/C47H73NO17/c1-27-17-15-13-11-9-7-5-6-8-10-12-14-16-18-34(64-46-44(58)41(48)43(57)30(4)63-46)24-38-40(45(59)60)37(54)26-47(61,65-38)25-33(51)22-36(53)35(52)20-19-31(49)21-32(50)23-39(55)62-29(3)28(2)42(27)56/h5-18,27-38,40-44,46,49-54,56-58,61H,19-26,48H2,1-4H3,(H,59,60)/b6-5+,9-7+,10-8+,13-11+,14-12+,17-15+,18-16+/t27-,28-,29-,30+,31+,32+,33-,34-,35+,36+,37-,38-,40+,41-,42+,43+,44-,46-,47+/m0/s1

(1R,3S,5R,6R,9R,11R,15S,16R,17R,18S,19E,21E,23E,25E,27E,29E,31E,33R,35S,36R,37S)-33-(((2R,3S,4S,5S,6R)-4-amino-3,5-dihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-1,3,5,6,9,11,17,37-octahydroxy-15,16,18-trimethyl-13-oxo-14,39-dioxabicyclo[33.3.1]nonatriaconta-19,21,23,25,27,29,31-heptaene-36-carboxylic acid

Amphotericin B;NSC 527017;Ambisome NSC527017;Amphozone FungilinFungizoneAMPH-B Fungizone Liposomal Amphotericin B NSC-527017

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO :~50 mg/mL (~54.11 mM)

Solubility in Formulation 1: 10 mg/mL (10.82 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 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.

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Solubility in Formulation 2: 10 mg/mL (10.82 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 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.

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