Light effect on the stability of β-lapachone in solution: Pathways and kinetics of degradation

Article abstract of DOI:10.1111/j.2042-7158.2011.01323.x

Objectives The purpose of this work was to study the chemical stability of the new antitumoral β-lapachone (βLAP) to determine the degradation pathway/s of the molecule and the degradation kinetics in addition to identifying several degradation products. Method Samples of βLAP in solution were stored under conditions of darkness and illumination at 40°C at which the pseudo-first order rate constants for the βLAP degradation were determined. Furthermore, drug degraded solutions were concentrated and purified using Sephadex LH-20 and preparative thin-layer chromatography and degradation products were identified by nuclear magnetic resonance spectroscopy. Key findings The results revealed that βLAP shows two different degradation routes: hydrolysis in the dark and photolysis under the light. The βLAP exposure to light accelerated the drug degradation about 140 fold, compared with the samples stored in the absence of light. The hydrolysis produced hydroxylapachol as the main degradation product. The photolysis yielded phthalic acid, 6-hydroxy-3methylene-3H-isobenzofuran-1-one and a benzomacrolactone together with a complex mixture of other phthalate-derivatives such as 2-(2-carboxy-acetyl)-benzoic acid. Conclusions This study provides useful information for the development of βLAP dosage forms, their storage, manipulation and quality control.

Full text of DOI:10.1111/j.2042-7158.2011.01323.x

Research Paper
JPP 2011, 63: 1156–1160
© 2011 The Authors
JPP © 2011 Royal
Pharmaceutical Society
Received November 12, 2010
Accepted May 30, 2011
DOI
Light effect on the stability of b-lapachone in solution:
pathways and kinetics of degradationjphp_1323 1156..1160
Marcílio S.S. Cunha-Filhoa, Ana Estévez-Braunb,c,
Elisa Pérez-Sacaub,c, Ma Magdalena Echezarreta-Lópezd,
Ramón Martínez-Pachecod and Mariana Landínd
10.1111/j.2042-7158.2011.01323.x
ISSN 0022-3573
aInstituto de Ciências da Saúde, Campus Universitário de Sinop, Universidade Federal de Mato Grosso
(UFMT), Avenida Alexandre Ferronato, Sinop, MT, Brazil, bInstituto Universitario de Bio-Orgánica ‘Antonio
González’, Universidad de La Laguna, La Laguna, Tenerife, cInstituto Canario de Investigación del Cáncer
(http://www.icic.es), La Laguna, Tenerife and dDepartamento de Farmacia y Tecnología Farmacéutica,
Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain
Abstract
Objectives The purpose of this work was to study the chemical stability of the new
antitumoral b-lapachone (bLAP) to determine the degradation pathway/s of the molecule
and the degradation kinetics in addition to identifying several degradation products.
Method Samples of bLAP in solution were stored under conditions of darkness and
illumination at 40°C at which the pseudo-first order rate constants for the bLAP degradation
were determined. Furthermore, drug degraded solutions were concentrated and purified
using Sephadex LH-20 and preparative thin-layer chromatography and degradation products
were identified by nuclear magnetic resonance spectroscopy.
Key findings The results revealed that bLAP shows two different degradation routes:
hydrolysis in the dark and photolysis under the light. The bLAP exposure to light accelerated
the drug degradation about 140 fold, compared with the samples stored in the absence of
light. The hydrolysis produced hydroxylapachol as the main degradation product. The
photolysis yielded phthalic acid, 6-hydroxy-3methylene-3H-isobenzofuran-1-one and a
benzomacrolactone together with a complex mixture of other phthalate-derivatives such as
2-(2-carboxy-acetyl)-benzoic acid.
Conclusions This study provides useful information for the development of bLAP dosage
forms, their storage, manipulation and quality control.
Keywords beta-lapachone; cancer chemotherapy; chemical photostability; degradation
products; preformulation
Introduction
b-Lapachone (bLAP) is a naphthoquinone obtained on a small scale from South
American trees of the Bigoniaceae and Verbenaceae families.[1] On a larger scale it can be
produced following the method developed by Hooker and co-workers[2] through cycliza-
tion of lapachol in sulfuric acid by an intramolecular nucleophilic attack of the oxygen
at C-4 on the carbocation of the isoprenyl side chain formed, and purified by further
recrystallization.
Over the last few years bLAP has been reported to possess a wide range of pharma-
cological properties, including antineoplastic activity, which has certainly generated
greater expectations from the molecule. In-vitro and in-vivo studies have shown that
bLAP inhibits conventional therapy-resistant tumours, particularly the proliferation of
neoplasms of slow cell cycles, like prostate, pancreatic, colon and lung cancer and some
ovarian and breast cancers.[3–9] Clinical trials on this molecule have recently been
launched.[10–12]
Correspondence: Mariana
Landin, Departamento de
Farmacia y Tecnología
Farmacéutica, Facultad de
Farmacia, Universidad de
Santiago de Compostela,
Campus Vida, Santiago de
Compostela 15782, Spain.
E-mail: m.landin@usc.es
It has been reported that bLAP is unstable under light irradiation;[13] however, as far as we
know, the degradation mechanism of bLAP is not yet fully understood and the kinetics of the
process have neither been established nor have the degradation products been identified,
which is important for manipulating formulations and for predicting possible side effects
and toxicity.
1156

b-Lapachone stability in solution
Marcílio S. S. Cunha-Filho et al.
1157
On this basis, the objective of this work is to study the dichloromethane–methanol (2 : 2 : 1) followed by preparative
stability properties of bLAP in solution both in the dark and TLC using dichloromethane–methanol (9 : 1) as eluent.
light under accelerated conditions, to determine the pathway/s
The identification of the isolated products was carried out
of degradation of the molecule, identify some degradation by proton nuclear magnetic resonance spectroscopy (1H
products and establish the kinetics of the processes.
NMR), carbon-13 nuclear magnetic resonance spectroscopy
(13C NMR), electron ionization mass spectrometry (EIMS)
and high-resolution electron ionization mass spectrometry
(HREIMS) analysis and by comparison with the data pub-
Materials and Methods
1
lished in chemical literature. H and 13C NMR spectra were
Materials
recorded in deuterated chloroform (CDCl3) on a Bruker
instrument at 300 and 75 MHz, respectively, with tetrame-
thylsilane (TMS) as the internal reference. High and low-
resolution mass spectra were obtained on a VG Autospec
spectrometer. Macherey-Nagel polygram Sil G/UV254 foils
were used for TLC and SIL G-100UV254 foils for preparative
TLC. Sephadex LH-20 was used for column chromatography.
bLAP (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-
5,6-dione; C15H14O3; MW 242.3) was supplied by the Labo-
ratorio Farmacêutico do Estado de Pernambuco/LAFEPE
(Recife, Brazil) with purity estimated by differential scanning
calorimetry (DSC) and high-performance liquid chroma-
tography (HPLC) to be 99.9%. Reference standard of
b-lapachone (083K1337) and lapachol (00509KN) were pur-
chased from Sigma-Aldrich (Steinhin, Germany). Methanol
for HPLC was obtained from Merck (Darmstadt, Germany).
The solutions were prepared using pure water (Millipore Milli
Q Plus quantification system; Massachusetts, USA). The sol-
vents and reagents used were of high-purity grade.
High-performance liquid chromatography
(HPLC) assay
bLAP assays were performed on a Waters M600 apparatus,
equipped with a C18 cartridge column (125 mm ¥ 45 mm ¥
5 mm) (Waters, Massachusetts, USA) based on a previously
reported method.[15] The mobile phase consisted of methanol–
water 65% (v/v). The isocratic flow rate was 1 ml/min at room
temperature. The injection volume was 20 ml. Chromato-
graphic detection was set at 253 nm with a photodiode array
detector. The bLAP test concentration was 40 mg/ml with a
retention time around 5 min. The mobile phase and samples
were filtered using a 0.45 mm nylon membrane (Waters,
USA). A system suitability test was evaluated by obtaining
the chromatographic parameters; capacity factor, number of
the theoretical plates and tailing factor.[16]
The validation of the analytical method was carried
out according to the ICH Q2.[17] Linearity in the range of
20–60 mm/ml was established (A = 112 967.C – 13 944; cor-
relation coefficient r = 0.9991 with degrees of freedom = 14,
F = 7078.5, a > 0.01). Precision and accuracy were studied,
the values being within the acceptable USP limits. The detec-
tion limit and quantification limit for the bLAP assay were
0.04 and 0.07 mg/ml respectively
Stability of b-lapachone in solution
bLAP aqueous solutions (30 mg/ml) prepared from a stock
ethanolic solution (20 mg/ml) were enclosed in glass
ampoules and stored at 40 Ϯ 2°C under the two conditions of
illumination (darkness and light). Light conditions were
established in accordance with option 2 of the International
Conference on Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH),[14] and
were applied using a modified oven equipped with two white
fluorescent lamps (Hitachi F8T5; Hitachi, Tokyo, Japan) and
two emission mercury UV lamps (370 nm) (Philips 08F8T5/
BLB; Philips, Eindhoven, Holland). Total brightness (1800
lx) and radiation UV (0.0078 W/m2) were measured by a
luximeter (HD9221; Delta Corps, Padua, Italy) equipped with
a probe (P912151). Both lamps were switched on simulta-
neously. The dark conditions were achieved by wrapping the
ampoules in foil and were included in the same environment.
Throughout one year, the percentage of the remaining
bLAP was determined in triplicate by HPLC at different
preset times. The logarithm of the percentage of the remaining
bLAP against time followed a linear trend making it possible
to calculate pseudo-first-order rate constants for the bLAP
degradation. Prediction of the drug shelf life was calculated
from the kinetics studied as the time that the drug retained
90% potency.
To obtain complete drug decomposition for validation pur-
poses and for purification of the bLAP degradation products,
samples of bLAP solution (30 mg/ml) were placed in an oven
at 50°C in light and darkness conditions until complete decom-
position. The selectivity of the analytical method for bLAP
against its photo and thermal degradation products was tested.
Results and Discussion
Purification and identification of the
degradation products of bLAP
bLAP stability in solution
The solutions of bLAP under degradation in the presence or Saturated aqueous solutions of bLAP present an intense
absence of light showed the formation of several compounds orange colour related to the naphthoquinone structure, which
upon TLC analysis. In the case of degradation under dark progressively became colourless as a consequence of expo-
conditions, the solvent was removed under reduced pressure sure to the light. However, the samples stored in darkness
and the corresponding residue was purified by preparativeTLC became red.
using dichloromethane–methanol (9 : 1) as the eluent. In the
The chromatograms of bLAP solutions before storage and
case of degradation under light conditions, the solvent was after storage in light and darkness are presented in Figure 1.
also removed under reduced pressure and the corresponding bLAP eluted at around 5 min under the HPLC conditions
residue was purified by Sephadex LH-20 column with hexane– selected in this study (Figure 1b). Irradiated bLAP solution

1158
Journal of Pharmacy and Pharmacology 2011; 63: 1156–1160
chromatograph (Figure 1a) showed the appearance of mul- degradation kinetic results. Drug shelf-life in the dark was 50
tiple peaks in the range of 0.9–3.8 min and the disappearance days but only 0.4 days if the drug is stored under light con-
of the bLAP peak. The chromatograph did not maintain the ditions. Light accelerated bLAP degradation about 140 fold,
bLAP peak in the sample stored in the dark (Figure 2c) and confirming the photo instability of this drug, as described in
presented two new earlier peaks at 0.9 and 1.6 min.
the literature.[13]
Table 1 summarizes data of the remaining drug during the
experiment under light and dark conditions as well as the ture, suggest the existence of two different degradation routes:
hydrolysis in the dark, and photolysis in the light.
These facts, together with the bLAP naphthoquinone struc-
(a)
Purification and identification of the
degradation products of bLAP
Aiming to investigate the degradation processes and identify
the main degradation products, we studied the crude materials
obtained after elimination of the solvent of the corresponding
solutions under light and dark conditions (Figure 2).
0.12
0.10
0.08
0.06
0.04
0.02
0.00
A dry sample of 71 mg stored in the dark was chro-
matographed on two preparative-TLC systems with
dichloromethane–methanol (9 : 1). Two products were
detected but just the major one could be isolated as an amor-
phous red wine solid (31 mg) and its molecular formula was
identified by high-resolution mass spectrometry (HRMS) as
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
(b)
0.40
0.30
0.20
0.10
0.00
1
C15H16O4. The main differences in the H NMR spectrum,
with respect to bLAP, were the chemical shifts of the aro-
matic and the aliphatic hydrogens. The 13C NMR spectrum
revealed the presence of a 1,4-naphthoquinone system
instead of 1,2-naphthoquinone moiety, as appears in bLAP.
All these data suggested that the product isolated was
hydroxylapachol (Figure 2, compound 2) which has been
previously obtained by synthesis from 2-hydroxy-1,4-
naphthoquinone[18] or from 2-hydroxy-3-(3-oxobutyl)-1,4-
naphthoquinone by reductive acetylation and treatment with
methyl magnesium iodide.[19]
Compound 2 has been identified as the only unconjugated
metabolite of bLAP in hepatocytes, a major metabolite in
dogs and a minor one in rats and humans.[20] This compound
showed low activity against carcinoma walker 256.[21]
13-Hydroxy-lapachol (2-hydroxy-3-(3-hydroxy-3-methyl-
butyl)-naphthalene-1,4dione): 1H NMR (CDCl3, 300 MHz) d:
8.05 (bs, 2H, H-5 + H-8), 7.69 (bs, 1H, H-6), 7.63 (bs, 1H,
H-7), 2.67 (bs, 2H, CH2-11), 1.68 (bs, 2H, CH2-12), 1.29
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
(c)
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Time (min)
Figure 1 HPLC chromatograms of b-lapachone solutions before
storage (b) and in completely degraded solutions after storage in light (a)
and darkness (c).
O
O
O
OH
Darkness
Light
OH
O
O
2
1
O
O
O
O
O
O
OH
OH
OH
COOH
Complex Mixture
of Phthalates
O
+
+
+
+
O H
O
O
O
3
O
5
O
6
4
Figure 2 Degradation routes of b-lapachone (1) under light and dark conditions. 2, Hydroxylapachol; 3, phthalic acid; 4, 6-hydroxy-3methylene-
3H-isobenzofuran-1-one; 5, (2-carboxy-acetyl)-benzoic acid; 6, benzomacrolactone.

b-Lapachone stability in solution
Marcílio S. S. Cunha-Filho et al.
1159
Table 1 Kinetics of b-lapachone in aqueous solution degradation at 40°C in light and dark conditions
Light
Darkness
Time (days)
Remaining drug %
(standard deviation)
Time (days)
0
14
32
80
105
166
250
Remaining drug %
(standard deviation)
0
3
4
5
6
7
8
10
100.0 (0.0)
56.7 (3.5)
43.2 (0.2)
27.7 (2.1)
23.7 (2.0)
18.9 (1.3)
12.1 (1.2)
6.2 (1.1)
280.0
100.0 (0.0)
99.5 (0.1)
95,6 (0.3)
87.0 (0.2)
84.2 (1.2)
73.8 (3.5)
60.4 (0.1)
51.8 (0.1)
2.1
320
Ka (10-3/day)
rb
Ka (10-3/day)
rb
0.9908
0.9979
Shelf-life (days)
0.4
Shelf-life (days)
50.0
b
aPseudo-first-order degradation rate constant. Correlation coefficient
(s, 6H, CH3-14 and 15). 13C NMR (CDCl3, 75 MHz) d: 184.7 been proposed to explain the Hooker’s oxidation mechanism
(C, C-4), 181.4 (C, C-1), 153.1 (C, C-2), 134.8 (CH, C-6), for 1,4-naphthoquinones.[24–26]
132.8 (C, C-10), 132.8 (CH, C-7), 129.5 (C, C-9), 126.6 (CH,
6-Hydroxy-3methylene-3H-isobenzofuran-1-one:
1H
C-5), 126.1 (CH, C-8), 124.7 (C, C-3), 71.0 (C, C-13), 41.5 NMR (MeOD, 300 MHz) d: 7.97 (d, 1H, 8.6 Hz), 7.38 (d, 1H,
(CH2, C-12), 29.1x2 (CH3, C-14 and 15), 18.3 (CH2, C-11). 2.6 Hz), 6.85 (dd, 1H, 8.6 and 2.6 Hz). 13C NMR (MeOD,
EIMS: 260 (1, M+), 245 (11, M+-Me), 242 (24, M+-H2O), 227 75 MHz) d: 160.1 (C), 134.2 (CH), 131.4 (C), 130.4 (C),
(100, M+-Me-H2O). HREIMS: 260.1055 (C15H16O4 calcd. 119.16 (CH), 117.4 (CH). EIMS: 164 (65, M+), 119 (100) 103
260.1049).
The solution of bLAP under light degradation conditions
(4) 91.(83) HREIMS: 164.0111 (C8H4O4 calcd. 164.0110).
1
The H NMR spectrum of the fourth product (Figure 2
1
was concentrated by the elimination of the solvent. The H compound 6) showed four signals corresponding to four aro-
NMR spectrum of the resulting crude material showed the matic hydrogens of a 1,2-disubstituted aromatic system,
existence of a complex mixture of phthalates. A dry sample of several complex signals for benzyl CH2 and aliphatic CH2, and
48 mg was first purified by a Sephadex LH-20 column. The two methyl groups. The 13C NMR analysis confirmed the
different fractions obtained were chromatographed on pre- presence of the 1,2-disubstituted aromatic ring and revealed
parative TLC. Four compounds were identified together the existence of various carbonyl groups. It showed a molecu-
with several inseparable mixtures of products (Figure 2, com- lar formula of C15H14O5, which seems to correspond with a
pounds 3, 4, 5 and 6). Three showed a retention factor (Rf) in benzomacrolactone as one of the less polar intermediates in
TLC lower than 0.1 using dichloromethane–methanol (9 : 1). the degradation of bLAP under light conditions.
1
This indicated the presence of polar functional groups in the
Benzomacrolactone: H NMR (CDCl3, 300 MHz) d: 8.22
corresponding structures. The first one showed a molecular (m, 1H), 8.05 (m, 1H), 7.81 (m, 2H), 3.03 (m, 1H), 2.29 (m,
formula of C8H5O4 in HRMS and two aromatic signals from 2H), 2.01 (m, 1H), 1.46 (s, CH3) 1.23 (s,CH3). 13C NMR
the 1H NMR and 13C NMR spectra. The analysis of these data (CDCl3, 75 MHz) d: 197.1 (C), 192.6 (C), 187.3 (C), 182.3
revealed that the isolated product was phthalic acid (Figure 2, (C), 134.9 (CH), 134.2 (CH), 131.4 (C), 130.1 (CH), 128.7
compound 3). The other compound (Figure 2, compound 4) (C), 126.4 (CH), 88.3 (C), 36.4 (CH2), 32.6 (CH2), 29.0 (CH3),
showed a similar EIMS spectrum to phthalic acid but it pre- 27.4 (CH3). EIMS: 260 (1, M+-CH2), 230 (9, M+-CO), 189
1
sented significant differences in the H NMR and the 13C (10) 104 149 (20) 132 (63) 104 (100) 76 (25) HREIMS:
NMR spectra. The most significant difference was the pres- 260.1065 (C15H16O4, M+-CH2, calcd 260.1049), 230.0944
ence of a three-substituted aromatic ring (d 7.97 (d, 1H, (C14H14O3, M+-CO, calcd. 230.0943).
8.6 Hz), 7.38 (d, 1H, 2.6 Hz), 6.85 (dd, 1H, 8.6 and 2.6 Hz)).
Isolated degradation products were dissolved in distilled
From these NMR data and the existence of a molecular ion water and qualitatively analysed by HPLC using the same
peak at m/z 164 in the EIMS spectra with the molecular conditions as previously described. Chromatograms of com-
formula C8H4O4 established by HREIMS, this product was pounds 2, 3, 4, 5 and 6 presented main peaks at shorter
identified as 6-hydroxy-3methylene-3H-isobenzofuran-1- retention times (0.9–3.8 min) than bLAP (4.9 min) suggesting
one.[22] Another compound isolated at a lower amount was that the degradation products did not interfere on bLAP
(2-carboxy-acetyl)-benzoic acid[23] (compound 5), which was resolution using the selected HPLC method. Moreover, UV
obtained as a mixture together with compound 4. The forma- spectra profiles of main peak of the compounds 2, 4 and 6
tion of these phthalic derivatives can be explained on the basis matched their corresponding peaks of the chromatograms
of an oxidative photolysis of bLAP. This process starts with of completely degraded solutions after storage in light
the oxidative cleavage of the C2-C3 bond to obtain a phthalic- (Figure 1a) and darkness (Figure 1c). Compound 3 is prob-
type intermediate, which suffers different radical decarboxy- ably overlapped and cannot be distinguished under the
lations to yield the other products. Similar compounds have conditions of the study.

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Journal of Pharmacy and Pharmacology 2011; 63: 1156–1160
7. Boorstein RJ, Pardee AB. Coordinate inhibition of DNA
synthesis and thymidylate synthase activity following DNA
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Conclusions
bLAP in solution showed two different degradation routes:
hydrolysis in the dark and photolysis in light. Hydrolysis
produced, as the main degradation product, hydroxyla-
pachol (2-hydroxy-3-(3-hydroxy-3-methyl-butyl)-[1,4]
naphthoquinone). Photolysis yielded phthalic acid, 6-hydroxy-
3methylene-3H-isobenzofuran-1-one, 2-(2-carboxy-acetyl)-
benzoic acid and a benzomacrolactone, together with a
complex mixture of other phthalate derivatives.
Light highly accelerates bLAP degradation in comparison
with dark conditions. These results provide useful information
for establishing the storage, manipulation and quality control
conditions for this drug.
10. Pardee AB et al. Cancer therapy with b-lapachone. Curr Cancer
Drug Targets 2002; 2: 227–242.
11. Khong HT et al. A phase 2 study of ARQ 501 in combination
with gemcitabine in adult patients with treatment naïve un-
resectable pancreatic adenocarcinoma. J Clin Oncol 2007; 25:
15017.
12. Hartner LP et al. Phase 2 dose multi-center, open-label study of
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tent, recurrent or metastatic leiomyosarcoma (LMS). J Clin
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Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest
to disclose.
Funding
14. ICH Q1B. Photostability testing of new drug substances and
products. International Conference on Harmonization, 1996.
15. Soares da Cunha Filho MS et al. Beta-lapachone: development
and validation of analytical method for the new therapeutic anti-
neoplastic alternative. Rev Bras Farm 2005; 86: 39–43.
16. USP 29. The United States Pharmacopoeia, 29th edn. Rockville,
MD: United States Pharmacopoeial Convention, 2006.
17. ICH Q2 (R1). Validation of analytical procedures: text and meth-
odology. International Conference on Harmonization, 2005.
18. Pettit GR, Houghton LE. Synthesis of hydroxyhydrolapachol
and lapachol. J Chem Soc 1971; 3: 509–511.
This work was supported by Xunta de Galicia
(PGIDIT008CSA007203PR) and the Programme Alban, the
European Union Programme of High Level Scholarships
for Latin America, scholarship No. E04D043994BR. A.E.B.
and E.P.S. thank the ICIC and the MCINN (SAF2009-
13296-C02-01) for financial support. M.L. thanks the Spanish
MEC for their financial support (PR2010-0460) during the
sabbatical year at Faculty of Science, University of Utrecht
(the Netherlands).
Acknowledgements
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Commn Mass Spectrom 2009; 23: 12–22.
Authors thank LAFEPE, Brazil, and Professor Dr Pedro Jose
Rolim Neto, Federal University of Pernambuco, Brazil, for
their kind gift of the bLAP and Ms J. Menis for her help in the
correction of the English version of the work.
21. Subramanian S, Ferreira MMC. A Structure-activity relationship
study of lapachol and some derivatives of 1,4-naphthoquinones
against carcinosarcoma Walker 256. Struct Chem 1998; 9:
47–57.
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