Peroral administration of beauverolides allows their transport into the peripheral blood and urine

Figures & data

Table 1. Overview of previously published beauverolide formulations.

Tabla 1. Resumen de las formulaciones de beauverólido publicadas anteriormente.

Compound	Solvent	Model	Reference
Vacuum-dried MeOH extracts of beauverolides – only compounds that dissolved in water (beauverolide La, beauverolide L, beauveriolide V, beauveriolide VI and likely some other compounds).	Water – an attempt to dissolve 10.9 g of crude extract in 700 mL of water led to selective solubilization of 1.1 g of the compounds	A549 cell line	(Park et al., 2018)
Beauveriolide III	Undisclosed	CHO cells	(Kapojos et al., 2018)
Beauveriolide-biotin-streptavidin-acrylamide beads	N/A	CHO-based microsomes	(Ohshiro et al., 2017)
Beauveriolide I and beauveriolide III	Undisclosed	Semi-intact (perforated) CHO cells and an intact ER fraction prepared from these cells	(Ohshiro et al., 2017)
Beauveriolide-biotin at 9.8 µM	Undisclosed	Mouse peritoneal macrophages	Suppl. mats. to Ohshiro et al. (2017)
Beauverolides C, F, I, Ja, L, M and N	Dissolved at 10 nM in DMSO; then added to 50 mM NaOAc [pH 5.1] and 10 μM CaCl2	In vitro calmodulin binding assay	Madariaga-Mazon et al. (2015)
Beauveriolide I	Dissolved in DMSO, then diluted 50-times in the culture medium to a final concentration of 20 μg mL^−1	Yeast survival	Nakaya et al. (2012)
Beauveriolides I and III	Dissolved in DMSO, then diluted to 0.1% v/v in DMSO and the compound at ≤5 μM	CHO cells	Witter et al. (2009)
Beauveriolides I and III, and their derivatives	Dissolved in MeOH, dilution 2.5 μL in 250 μL of medium; final concentrations of the compounds were ≤20 – ≤40 μM	CHO cells	Ohshiro et al. (2009)
Beauveriolides I and III–XIII (i.e. incl. beauverolides M and Fa)	≤10 μM	Mouse peritoneal macrophages; microsomes prepared from mouse macrophages; microsomes from mouse liver	Namatame et al. (1999a); Matsuda et al. (2004); Tomoda and Doi (2008)
Beauveriolide III and its derivatives. Among them, D-Ala analogues consisting of A{2} improved solubility (the authors did not disclose any details)	Undisclosed	Mouse peritoneal macrophages; mouse liver microsomes	Nagai et al. (2008)
Beauveriolides I and III	Dissolved in MeOH, dilution 2.5 μL in 250 μL (or 2 μL in 200 μL) of medium; for experiments with microsomes, 10 μL in 200 μL of medium; final concentrations of the compounds were ≤20 μM	Mouse peritoneal macrophages; microsomes; CHO cells	Ohshiro et al. (2007)
Stereoisomers of beauveriolide III	Dissolved in MeOH, dilution 2.5 μL in 250 μL of medium; final concentrations of the compounds were ≤20 – ≤52.1 μM	Mouse peritoneal macrophages; CHO cells	Ohshiro et al. (2006)
Beauveriolides I and III, and their derivatives	Undisclosed; final concentrations of the compounds were ≤20 μM	Mouse peritoneal macrophages	Nagai et al. (2006)
Beauveriolide III	0.15% cholesterol-supplemented diet was supplemented with beauveriolide III (50 or 25 mg kg^−1 of body weight) suspended in 0.05% sodium carboxymethylcellulose, p.o., every day for 2 months	apoE-KO and LDLR-KO mice (in vivo)	Namatame et al. (2004)
Beauveriolides I and III	Dissolved in MeOH, dilution 2.5 μL in 250 μL of medium or 10 μL in 200 μL of medium; final concentrations of the compounds were ≤100 μM	Mouse peritoneal macrophages; mouse liver microsomes or membranes, mouse liver, Caco2 cells	Namatame et al. (2004)
Beauveriolides I and III	Insoluble in H2O and n-hexane; soluble in EtOH, CH3CN, CH3OH, CHCl3, and EtOAc	N/A	Namatame et al. (1999a)
Beauveriolides I and III	Dissolved in MeOH, dilution 1.25 μL in 125 μL of medium; final concentrations of the compounds were ≤100 μM used at ≤25 μM in solutions, effective concentrations were 3 μM or 10 μM (macrophages); undisclosed, effective concentration 2.1 mM (1 mg mL^−1, and 10 μg per disc) (microbes); dissolved directly in the medium containing 0.01% egg lecithin, 7.5 mM NaHCO3, 7.5 mM KCl, 7.5 mM CaCl2 · 2H2O, and 7.5 mM MgSO4 · 7H2O, effective concentration 0.2 mM (100 μg mL^−1) (nematodes, insects)	Mouse peritoneal macrophages; various microbes; nematode (Caenorhabditis elegans) and insects (Artemia salina)	Namatame et al. (1999b)
Beauveriolides IV– IX	Insoluble in H2O and n-hexane; soluble in EtOH, CH3CN, MeOH, CHCl3, and EtOAc; used in ≤25 μM solutions (macrophages) or applied at 2 mM concentration to the discs to a final concentration 10 μg per disc (microbes) or used at 0.2 mM (100 μg mL^−1) (nematodes, insects)	Mouse peritoneal macrophages; various microorganisms; unspecified nematodes and insects	Matsuda et al. (2004)
Beauverolide L	Dissolved in EtOH at 10 mg mL^−1; diluted with sterile saline to 1 mg mL^−1. Ten ml of this preparation was injected per larva (10 mg toxin per larva).	Galleria mellonella larvae	Vilcinskas et al. (1999)
Beauverolide L	Metabolite-coated particles were used as 10% (w:v) suspension in sterile linseed oil. The larvae were injected with 10 ml of suspension, corresponding to a final beauverolide concentrations of 10–30 mg per larva	Galleria mellonella larvae	Vilcinskas et al. (1999)

Abbreviations: apoE – apolipoprotein E; CHO – chinese hamster ovary; DMSO – dimethyl sulfoxide; ER – endoplasmic reticulum; EtOAc – ethyl acetate; EtOH – ethanol; LDLR – low density lipoprotein receptor; MeOH – methanol; NaOAc – sodium acetate

Abreviaturas: apoE – apolipoproteína E; CHO – ovario de hámster chino; DMSO – dimetilsulfóxido; ER – retículo endoplásmico; EtOAc – acetato de etilo; EtOH – etanol; LDLR – receptor de lipoproteínas de baja densidad; MeOH – metanol; NaOAc – acetato de sodio

Table 2. Overview of tested solubilization conditions for the beauverolides.

Tabla 2. Resumen de las condiciones de solubilización probadas para los beauverólidos.

Solvent	Solubility limit and solubilization conditions	Conditions under which the formation of gels or precipitates occurs
DMSO
● 80%		1 mg mL^−1 (60°C and 72°C)
EtOH
● 96%	2 mg mL^−1 (72°C)	2 mg mL^−1 (4°C)
● 50% (obtained from 75% EtOH by evaporation)		2 mg mL^−1 (72°C – precipitation; 20°C – gel formation)
EtOH in Tris, pH 7.4
● 75% in 200 mM Tris, pH 7.4	2 mg mL^−1 (72°C, 37°C – 5 h); 1 mg mL^−1 (72°C, 37°C – 4 h, O.N.; 20°C – 4 h); 0.75 mg mL^−1 (37°C – O.N.; 20°C – 4 h); 0.5 mg mL^−1 (37°C – O.N.; 20°C – 4 h); 0.25 mg mL^−1 (37°C – O.N.; 20°C – 4 h)	2 mg mL^−1 (20°C O.N. – gel formation); 1 mg mL^−1 (20°C O.N. – gel formation); 0.75 mg mL^−1 (20°C O.N. – gel formation); 0.5 mg mL^−1 (20°C O.N. – gel formation)
● 75% in 100 mM Tris, pH 7.4	2 mg mL^−1 (72°C, 37°C – 5 h); 1 mg mL^−1 (72°C, 37°C – 4 h, O.N.; 20°C – 4 h); 0.75 mg mL^−1 (37°C – O.N.; 20°C – 4 h); 0.5 mg mL^−1 (37°C – O.N.; 20°C – 4 h); 0.25 mg mL^−1 (37°C – O.N.; 20°C – 4 h)	2 mg mL^−1 (20°C O.N. – gel formation); 1 mg mL^−1 (20°C O.N. – gel formation); 0.75 mg mL^−1 (20°C O.N. – gel formation); 0.5 mg mL^−1 (20°C O.N. – gel formation)
● 75% in 50 mM Tris, pH 7.4	2 mg mL^−1 (72°C, 37°C 5 h); 1 mg mL^−1 (72°C, 37°C 3 h, 4 h, O.N.; 20°C 4 h); 0.75 mg mL^−1 (37°C O.N.; 20°C 4 h); 0.5 mg mL^−1 (72°C; 37°C 3 h, O.N.; 20°C 4 h, O.N.); 0.25 mg mL^−1 (72°C; 37°C 3 h, O.N.; 20°C 4 h, O.N.); 0.125 mg mL^−1 (72°C; 37°C 3 h; 20°C O.N.; 4°C O.N.); 62.5 μg mL^−1 (72°C; 37°C 3 h; 20°C O.N.; 4°C O.N.); 31.3 μg mL^−1 (72°C; 37°C 3 h; 20°C O.N.; 4°C O.N.); 15.6 μg mL^−1 (72°C; 37°C 3 h; 20°C O.N.; 4°C O.N.)	2 mg mL^−1 (37°C – 3 h; 20°C; 4°C O.N. – gel formation); 1 mg mL^−1 (20°C O.N. – gel formation; 4°C O.N. – gel formation); 0.75 mg mL^−1 (20°C O.N. – gel formation); 0.5 mg mL^−1 (20°C O.N. – gel formation; 4°C O.N. – gel formation); 0.25 mg mL^−1 (4°C O.N. – gel formation); 0.125 mg mL^−1 (4°C for a week – gel formation)
● 75% in 25 mM Tris, pH 7.4	2 mg mL^−1 (72°C, 37°C – 5 h); 1 mg mL^−1 (72°C, 37°C – 4 h, O.N.; 20°C – 4 h); 0.75 mg mL^−1 (37°C – O.N.; 20°C – 4 h); 0.5 mg mL^−1 (37°C – O.N.; 20°C – 4 h); 0.25 mg mL^−1 (37°C – O.N.; 20°C – 4 h)	2 mg mL^−1 (20°C O.N. – gel formation); 1 mg mL^−1 (20°C O.N. – gel formation); 0.75 mg mL^−1 (20°C O.N. – gel formation); 0.5 mg mL^−1 (20°C O.N. – gel formation)
● 60% in 200 mM Tris, pH 7.4		1 mg mL^−1 (72°C; 20°C – gel formation)
● 60% in 100 mM Tris, pH 7.4		1 mg mL^−1 (72°C; 20°C – gel formation)
● 60% in 50 mM Tris, pH 7.4	0.2 mg mL^−1 (72°C, 37°C – O.N.); 0.1 mg mL^−1 (72°C, 37°C – O.N.; 20°C – 6 h); 50 μg mL^−1 (72°C, 37°C – O.N.; 20°C – 6 h); 25 μg mL^−1 (72°C, 37°C – O.N.; 20°C – 6 h; 4°C – O.N.)	1 mg mL^−1 (72°C; 20°C – gel formation); 0.2 mg mL^−1 (20°C – 6 h); 0.1 mg mL^−1 (4°C – O.N.); 50 μg mL^−1 (4°C – O.N.)
● 60% in 25 mM Tris, pH 7.4		1 mg mL^−1 (72°C; 20°C – gel formation)
EtOH in phosphate buffer, pH 7.4
● 75% in phosphate buffer pH 7.4 (200 mM, 100 mM, 50 mM, or 25 mM, respectively)	2 mg mL^−1 (72°C); 1 mg mL^−1 (37°C – O.N.)	2 mg mL^−1 (37°C); 1 mg mL^−1 (20°C – gel formation)
EtOH with trehalose
● 75% EtOH + 2%, 1%, 0.5%, or 0.25% trehalose	2 mg mL^−1 (72°C)	2 mg mL^−1 (37°C 2 h); 1 mg mL^−1 (37°C O.N.; 20°C – gel formation)
● 40% EtOH + 4%, 2%, 1% or 0.5% trehalose (obtained from 75% EtOH by evaporation)	2 mg mL^−1 (72°C)	2 mg mL^−1 (37°C – gel formation; 20°C – solid gel); 1 mg mL^−1 (37°C – gel formation; 20°C – solid gel); 0.7 mg mL^−1 (37°C – gel formation; 20°C – solid gel); 0.5 mg mL^−1 (37°C – gel formation; 20°C – gel formation); 0.25 mg mL^−1 (37°C – gel formation; 20°C – gel formation); 0.125 mg mL^−1 (37°C – gel formation; 20°C – gel formation)
Kolliphor EL
● 50% (v/v) Kolliphor EL diluted in 96% EtOH		8 mg mL^−1 (60°C – solid gel; 20°C – solid gel); 3 mg mL^−1 (60°C – solid gel; 20°C – solid gel);
● 25% (v/v) Kolliphor EL diluted in 96% EtOH	2 mg mL^−1 (60°C, 37°C – 4 hod)	2 mg mL^−1 (37°C O.N.; 20°C – solid gel);
● 10% (v/v) Kolliphor EL diluted in 96% EtOH	2 mg mL^−1 (60°C, 37°C – 4 hod)	2 mg mL^−1 (37°C O.N.; 20°C – solid gel);
● 5% (v/v) Kolliphor EL diluted in 96% EtOH	2 mg mL^−1 (60°C, 37°C – 4 hod)	2 mg mL^−1 (37°C O.N.; 20°C – solid gel);
● 12.5% (v/v) Kolliphor EL, 37.5% (v/v) EtOH, 0.45% (m/v) NaCl		1 mg mL^−1 (solid gel);
● 5% (v/v) Kolliphor EL, 37.5% (v/v) EtOH, 0.45% (m/v) NaCl		1 mg mL^−1 (solid gel);
● 2.5% (v/v) Kolliphor EL, 37.5% (v/v) EtOH, 0.45% (m/v) NaCl		1 mg mL^−1 (solid gel);
● 12.5% (v/v) Kolliphor EL, 50% (v/v) EtOH, 50 mM Tris, pH 7.4	0.5 mg mL^−1 (72°C)	0.5 mg mL^−1 (37°C – O.N.; 20°C – precipitate);
● 5% (v/v) Kolliphor EL, 50% (v/v) EtOH, 50 mM Tris, pH 7.4	0.5 mg mL^−1 (72°C)	0.5 mg mL^−1 (37°C – O.N.; 20°C – solid gel after three days);
● 2.5% (v/v) Kolliphor EL, 50% (v/v) EtOH, 50 mM Tris, pH 7.4	0.5 mg mL^−1 (72°C)	0.5 mg mL^−1 (37°C – O.N.; 20°C – gel formation after three days)

The experiments were targeted at the solubilization of a mixture of beauverolides with predominant beauverolide I at 2 mg mL⁻¹.

Abbreviations: DMSO – dimethyl sulfoxide; EtOH – ethanol; O.N. – overnight

Los experimentos tuvieron por objeto examinar la solubilización de una mezcla de beauverólidos compuesta predominantemente de beauverólido I a 2 mg mL⁻¹.

Abreviaturas: DMSO – dimetilsulfóxido; EtOH – etanol; O.N. – nocturno

Table 3. Orally administered beauverolides from gel capsules are detectable in serum and excreted in intact form by urination.

Tabla 3. Los beauverólidos administrados oralmente mediante cápsulas de gel son detectables en el suero y son excretados intactos al orinar.

			Input (BC-BVD103)		Urine (formulation without additives)		Urine (formulation with Nutrilon)		Urine (formulation with glucose)		Serum (formulation without additives)		Serum (formulation with Nutrilon)		Serum (formulation with glucose)
Beauverolide	[MW]	[M + H]^+	RT	Relative content [%]	RT	Relative share [%]	RT	Relative share [%]	RT	Relative share [%]	RT	Relative share [%]	RT	Relative share [%]	RT	Relative share [%]
N	503.4	504.3068	1.8	0.79		0.00	1.78	1.49	1.78	1.95		0.00		0.00		0.00
M	439.4	440.3119	2.23	16.15	2.22	38.18	2.21	35.89	2.19	41.92	2.20	15.94	2.19	22.88	2.18	33.96
Q	526.4	527.3228	2.44	7.73	2.44	2.22	2.40	3.38	2.40	2.91	2.50	0.85	2.40	3.71		0.00
I	487.4	488.3119	2.61	34.94	2.61	44.31	2.58	50.15	2.58	34.19	2.57	28.57	2.59	52.33	2.57	36.45
P	467.4	468.3432	3.23	3.76	3.20	4.13	3.19	2.41	3.17	11.42		0.00		0.00	3.16	21.62
E	515.4	516.3432	3.35	4.57	3.34	4.79	3.30	3.47	3.28	2.36	3.29	54.63	3.31	2.16	3.30	1.59
J	602.4	603.3541	3.48	0.97		0.00		0.00		0.00		0.00		0.00		0.00
R	554.4	555.3541	3.5	1.38		0.00		0.00		0.00		0.00		0.00		0.00
L	563.4	516.3432	3.78	8.00	3.75	6.38	3.73	3.21	3.71	5.21		0.00	3.71	6.86	3.70	6.37
F	515.4	564.3432	3.86	17.57		0.00		0.00		0.00		0.00	3.79	12.06		0.00
B	543.4	544.3745	4.81	0.16		0.00		0.00		0.00		0.00		0.00		0.00
K	630.4	631.3854	4.73	0.97		0.00		0.00	4.65	0.05		0.00		0.00		0.00
C	591.4	592.3745	5.33	3.03		0.00		0.00		0.00		0.00		0.00		0.00
Total beauverolides [ng ml^−1]						33.69		11.25		147.66		8.10		9.45		55.17
Estimated total beauverolides per mouse [ng]						25.27		8.44		110.74		9.73		11.34		66.20
Estimated share of applied beauverolides [%]						1.7 * 10^−3		0.6 * 10^−3		7.4 * 10^−3		0.7 * 10^−3		0.8 * 10^−3		4.4 * 10^−3

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