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Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids.

ABSTRACT

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency ([IC.sub.50] < 10 [micro]M) and 15/36 inhibiting CYP3A4 potently ([IC.sub.50] < 1 [micro]M). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.

[C] 2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Corydalis cava

Papaveraceae

Isoquinoline alkaloids

Cytochrome P450(CYP)

Inhibition

Fluorometric assay

Introduction

Plant alkaloids, one of the largest groups of natural products, represent a highly diverse group of chemical entities. Plants are estimated to produce approximately 12,000 different alkaloids with a wide range of pharmacological properties. Alkaloids can be classified according to their basic heterocyclic ring system. Benzylisoquinoline alkaloids are a group of nitrogen-containing plant secondary metabolites of which approximately 2500 members have been identified. Many of these compounds possess potent pharmacological effects. For example, the well known plant alkaloids include the narcotic analgesics, morphine and codeine, apomorphine (a derivative of morphine) used in Parkinson's disease, the muscle relaxant papaverine, and the antimicrobial agents sanguinarine and berberine. Also several potent anti-cancer drugs have been developed from plant compounds (Stevigny et al. 2005; Ziegler and Facchini 2008; Ziegler et al. 2009). In addition, several Stephania and Corydalis species are used in Traditional Chinese Medicine (TCM) because of their alkaloid content (Mo et al. 2007).

The cytochrome P450 (CYP) enzymes constitute a superfamily of heme-containing mono-oxygenases that catalyse the oxidative metabolism of a wide variety of xenobiotics, including drugs, plant-derived or fungal-derived secondary metabolites consumed with food, and a large number of environmental pollutants, industrial compounds, herbicides, and pesticides. The human CYP forms that metabolize xenobiotics belong to the families CYP1, CYP2 and CYP3. Individual CYP enzymes in these families have broad and overlapping substrate specificities, and are responsible for the metabolism of approximately 70-80% of all currently used drugs (Nebert and Russell 2002; Guengerich et al. 2005; Ingelman-Sundberg 2005). CYP enzymes also play a key role in oxidative reactions in plant secondary metabolism (Ziegler and Facchini 2008), and thus plants have remarkably high numbers of CYP genes (Nielsen and Moller 2005).

To a large extent metabolism determines the pharmacokinetic behaviour of a drug, i.e., the intensity and the duration of action. Modulation of CYP activity via inhibition or induction by drugs and other xenobiotics often is the source of drug interactions. Drug interactions can evoke severe adverse effects, they have resulted in early termination of drug development, refusal to obtain approval, severe prescribing restrictions, and even withdrawal of drugs from the market. The most common mechanism of drug interactions is inhibition of these CYP enzymes (Kalgutkar et al. 2007; Pelkonen et al. 2008). On the other hand, specific CYP inhibitors that do not interact with other targets and have a clear pharmacodynamic profile are potential co-therapeutics, especially in the field of antivirals. The so-called pharmacokinetic boosters inhibit the CYPs metabolizing antiviral drugs, e.g. the HIV-1 protease inhibitor lopinavir, is used to raise the effective concentration of the antiviral drug in the body. The advantages of this approach range from possible reductions in drug load to improvements of patient compliance by achieving longer dosing intervals (Dickinson et al. 2010).

In early drug development, experiments are routinely carried out to determine which CYP enzymes catalyse the metabolism of lead compounds. In addition, the potential of lead compounds to inhibit CYPs can be evaluated with in vitro methods. Often, but not always, a compound that inhibits a specific CYP form is also a substrate for that same form. These experiments employ various sources of CYP enzymes, e.g. human liver or cDNA-expressed human enzymes, and probe substrates and inhibitors (Bjornsson et al. 2003; van de Waterbeemd and Gifford 2003; Pelkonen and Raunio 2005).

There is still plenty of scope to enlarge the known chemical space of CYP inhibitors and substrates. The purpose of this study was (1) to screen a series of novel plant-derived isoquinoline alkaloids for their abilities to interact with the most important human liver xenobiotic-metabolizing CYP enzymes, and (2) to search for novel CYP form-selective inhibitory compounds. The inhibition potency of eight protopine, six aporphine, one spirobenzylisoquinoline and one phthalideisoquinoline, and 20 protoberberine alkaloids was determined against recombinant CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes.

Materials and methods

Chemicals and reagents

The origin of the tested alkaloids is the following (numbering according to Table 2). Alkaloid 1 was isolated by W. H. Perkin in 1918 from Bocconia cordata Willd. Alkaloid 2 was isolated by J. Slavik in 1966 from Hunnemannia fumariifolia Sweet. Alkaloid 3 was isolated by W. H. Perkin in 1926 from Papaver somniferum L. Alkaloid 4 was isolated by J. Slavik from Chelidonium majus L. Alkaloid 27 was isolated by J. Slavik from Meconopsis cambrica (L.) Vig. Alkaloids 5, 6, 8-14 and 19-26 were from a historical collection gathered by Johannes Gadamer and co-workers at the beginning of the 20th century. Alkaloid 8 thereof is the semisynthetic derivative of 9. Alkaloids 5, 6, 9-12, 14 and 19-26 were isolated from Corydalis cava L. and alkaloid 13 from Fumaria vaillantii Loisel. Alkaloids 15-18 are semisynthetic and alkaloids 28-36 synthetic (Meyer et al. unpublished). Alkaloid 7 was purchased from Sigma Aldrich (St. Louis, MO, USA). The purity of the alkaloids was higher than 95% as determined by gas chromatography-mass spectrometry and combustion analysis. All structures were confirmed by [1.sup.H] NMR and mass spectrometry.

cDNA-expressed human wild-type CYPs (Supersomes[TM]) were purchased from BD Biosciences Discovery Labware (Bedford, MA, USA). Substrates and metabolite standards were purchased from BD Biosciences Discovery Labware (Bedford, MA) and Sigma Chemical Company (St. Louis, MO). These were 7-ethoxyresorufin and resorufin for CYP1A2; coumarin and 7-hydroxycoumarin for CYP2A6; 7-ethoxy-4-(trifluoromethyl) coumarin (EFC) and 7-hydroxy-4-(trifluoromethyl) coumarin (HFC) for CYP2B6; dibenzylfluorescein (DBF) and fluorescein for CYP2C8 and CYP2C19; 7-methoxy-4-(trifluoromethyl) coumarin (MFC) and HFC for CYP2C9 and CYP3A4; and 7-methoxy-4-(aminomethyl) coumarin (MAMC) and 7-hydroxy-4-(aminomethyi) coumarin (HAMC) for CYP2D6. All other chemicals used were from Sigma-Aldrich (St. Louis, MO, USA) and were of the highest purity available.

CYP inhibition assays

Incubations were conducted in a 150 [micro]l volume in 96-well microtiter plates based on the general principles originally published by Crespi et al. (1997), using cDNA-expressed recombinant CYP enzymes. Each test compound was screened using four concentrations (mainly 1:10 ratio) ranging from 0.01 to 1000 [micro]M in a duplicate layout. The incubation mixtures contained l00 mM Tris/HCl buffer (pH 7.4), 0.75-2.0 pmol of CYP enzyme, the probe substrate at the concentration corresponding to its measured apparent [K.sub.m], and the nicotine adenine dinucleotide phosphate hydrogen (NADPH)-regenerating system (consisting of 1.13 mM NADP, 12.5 mM isocitric acid, 56.33 mM KCl, 187.5 mM Tris/HCl, pH 7.4, 12.5 mM [MgCl.sub.2], 0.0125 mM [MnCl.sub.2], 0.075 U [ml.sup.-1] isocitrate dehydrogenase), except 50 mM Tris/HCl buffer (pH 7.4), 0.3 mM NADPH, and 5 mM [MgCl.sub.2] in a l00 [micro]l incubation volume in the case of CYP2A6. Due to their high lipophilicity the test compounds were dissolved in acetonitrile (ACN) or dimethyl sulfoxide (DMSO) and then further diluted with water. Consequently, the final solvent concentrations in the incubations did not exceed 2%. Only the hydrochloride salts were directly soluble in water. Controls were treated similarly but without the presence of inhibitors. The reactions were initiated by adding 50 [micro]l of the NADPH-regenerating system, except for 25 [micro]l of 1.2 mM NADPH in the case of CYP2A6, after a 10-min preincubation at 37[degrees]C. After incubation (10-60 min), the reactions were terminated by addition of the stop solution (Table 1). In addition, in the case of CYP2A6, immediately before the measurement, 140 [micro]l of 1.6 M glycine-NaOH buffer (pH 10.4) was added into the wells. The experimental conditions are summarized in Table 1.
Table 1

Experimental conditions in human recombinant CYP enzyme inhibition
assays.

CYP   Substrate ([mu]M)  Fluorescent        Incubation  Enzyme
                        metabolite         time (min)  (pmol)

1A2   7-Ethoxyresorufin  Resorufin          20          0.5
     (1)

2A6   Coumarin(10)       7-Hydroxycoumarin  10          0.3

2B6   EFC (2.5)          HFC                30          0.75

2C8   DBF (0.5)          Fluorescein        30          1.5

2C9   MFC (75)           HFC                45          1.5

2C19  DBF (0.5)          Fluorescein        30          1.5

2D6   MAMC (20)          HAMC               60          2.0

3A4   MFC (50)           HFC                30          1.0

CYP   Excitation/emission
     (nm)

1A2   570/615

2A6   355/460

2B6   405/535

2C8   485/535

2C9   405/535

2C19  485/535

2D6   405/460

3A4   405/535

Abbreviations: EFC. 7-ethoxy-4-(trifluoromethyl)coumarin; DBF,
dibenzylfluorescein; MFC. 7-methoxy-4-(trifluoramethyl)coumarin; BFC.
7-benzyloxy-4-(trifluorumethyl)coumarin; MAMC, 7-methoxy-
4-(aminomethyl)coumarn; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin;
HAMC, 7-hydroxy-4-(aminomethyl)coumarin.


Fluorescence was measured with a Victor (2) plate counter (Perkin-Elmer Life Sciences Wallac, Turku, Finland) with excitation and emission wavelengths set according to the probe substrate/metabolite. The [IC.sub.50] values (inhibitor concentration that reduced the metabolism of the CYP probe substrate by 50%) were calculated using non-linear regression analysis with Prism 4.0 software (San Diego, CA, USA).

The linearity of the reactions with respect to incubation time and protein concentration as well as the basic kinetic parameters ([K.sub.m] and [V.sub.max]) for the probe substrates was determined before the actual experiments (data not shown). In addition, several control incubations (with positive controls) were carried out to confirm the reliability of the assays, as described previously (Turpeinen et al. 2006). The extent of inhibition was classified as potent ([IC.sub.50] < 1 [micro]M), marginal/moderate (1 [micro]M < [IC.sub.50] < 10 [micro]M), weak ([IC.sub.50] > 10 [micro]M), or no inhibition ([IC.sub.50] > 100 [micro]M) (White 2000).

Results

The capabilities of plant isoquinoline alkaloids to inhibit individual CYP enzymes, i.e., changes caused by the alkaloids in the metabolism of CYP-specific probe substrates, were determined with eight recombinant human CYPs. The alkaloids were classified to four groups according to their inhibition potency: potent [IC.sub.50] < 1 [micro]M, marginal/moderate 1 [micro]M < [IC.sub.50] < 10 [micro]M, weak [IC.sub.50] < 10 [micro]M, or no inhibition [IC.sub.50] > 100 [micro]M (White 2000). The screening results for all alkaloids and CYP forms are presented in Table 2. A summary of the number and the percentage of alkaloids in each inhibition potency category are provided in Table 3. The results revealed that the majority of alkaloids inhibited most potently the CYP3A4 form (30/36 with at least moderate potency and 15/36 potently), and CYP2D6 (26/34 with at least moderate potency and 21/34 potently), and to some extent also CYP2C19 (15/36 with at least moderate potency and 3/36 potently). The alkaloids had a clearly less inhibitory effect on CYP2C8 (3/36 with moderate potency), CYP2B6 (4/36 with moderate potency), and CYP2C9 (6/36 with moderate potency). Furthermore, 10/36 alkaloids inhibited CYP1A2 with moderate potency and 1/36 (nantenine) potently. The tested alkaloids did not inhibit CYP2A6.
Table 2

CYP inhibition screening results organized according to the
isoquinoline alkaloid subgroups.

No.  Compound                                   1A2  2A6  2B6  2C8  2C9

1    Protopine                                  ++   -    +    -    -

2    Hunnemannine                               +    -    +    ++   -

3    Cryptopine                                 -    -    -    +    -

4    Allocryptopine                             +    -    +    +    -

5    Corycavidine                               -    -    +    +    -

6    Corycavamine                               +    -    +    +    -

15   Dihydrocryptopine                          +    -    -    +    -

16   Dihydroprotopine                           +    -    ++   +    ++

7    Apomorphine HCl                            ++   -    +    ++   ++

8    Bulbocapninemethylether                    ++   -    +    -    +

9    Bulbocapnine                               ++   -    +    -    +

10   Isocorydine                                +    -    +    -    +

11   Corydine                                   +    -    +    -    +

12   Nantenine                                  +++  -    +    ++   ++

13   Parfumine                                  +    -    +    -    +

14   Capnoidine                                 +    -    -    +    -

17   Canadine                                   ++   -    ++   -    -

18   Nandinine                                  ++   -    ++   +    +

19   Corydaline                                 +    -    -    +    ++

20   Tetrahydropalmatine                        *    -    -    -    +

21   Corypalmine                                +    -    -    +    +

22   Stylopine                                  ++   -    +    -    ++

23   Thalictricavine                            ++   -    ++   +    -

24   Isocorybulbine                             +    -    -    +    +

25   Corybulbine                                +    +    -    +    +

26   Scoulerine HCl                             +    -    +    +    +

27   Mecambridine                               +    -    -    +    +

28   2,3,10,11 -Tetramethoxyberbin              +    -    -    -    -

29   8-Ethyl-2,3,10,ll-retramethoxyberbine      +    -    +    -    -

30   8-Methyl-2,3,10,11 -tetramethoxyberbine    +    -    -    -    ++

31   8-Methyl-2,3,10,11-retraethoxyberbine      +    -    +    +    -

32   2,3-Dimethoxy-10,11-methylenedioxyberbine  +    -    -    -    -

33   2,3-Dimethoxy-9,10-methylenedioxyberbine   +    -    +    +    +

34   3,10,11-Trimethoxyberbine                  +    -    +    -    -

35   3-Methoxyberbine                           ++   -    +    -    -

36   2.3-Dimethoxyberbine HC1                   ++   -    -    -    +

No.  Compound                                   2C19  2D6  3A4

1    Protopine                                  ++    +++  +

2    Hunnemannine                               +     +++  ++

3    Cryptopine                                 ++    +++  -

4    Allocryptopine                             -     +++  ++

5    Corycavidine                               +     ++   ++

6    Corycavamine                               ++    +++  ++

15   Dihydrocryptopine                          ++    ++   +++

16   Dihydroprotopine                           +++   +++  +++

7    Apomorphine HCl                            +     +    +

8    Bulbocapninemethylether                    +     ++   +++

9    Bulbocapnine                               +     ++   +++

10   Isocorydine                                +     +    ++

11   Corydine                                   +     +    +++

12   Nantenine                                  ++    ND   +++

13   Parfumine                                  +     +    +++

14   Capnoidine                                 +++   +++  +++

17   Canadine                                   ++    +++  +++

18   Nandinine                                  ++    +++  ++

19   Corydaline                                 ++    +    +++

20   Tetrahydropalmatine                        +     +++  ++

21   Corypalmine                                ++    ++   +++

22   Stylopine                                  +++   +++  +++

23   Thalictricavine                            +     ND   +

24   Isocorybulbine                             -     +    ++

25   Corybulbine                                +     -    ++

26   Scoulerine HCl                             +     +++  ++

27   Mecambridine                               -     +++  ++

28   2,3,10,11 -Tetramethoxyberbin              -     +++  ++

29   8-Ethyl-2,3,10,ll-retramethoxyberbine      -     +++  ++

30   8-Methyl-2,3,10,11 -tetramethoxyberbine    +     +++  ++

31   8-Methyl-2,3,10,11-retraethoxyberbine      +     +    +++

32   2,3-Dimethoxy-10,11-methylenedioxyberbine  ++    +++  ++

33   2,3-Dimethoxy-9,10-methylenedioxyberbine   +     +++  +++

34   3,10,11-Trimethoxyberbine                  ++    +++  +++

35   3-Methoxyberbine                           ++    +++  -

36   2.3-Dimethoxyberbine HC1                   +     +++  +

+++, [IC.sub.50] < 1 [micro]M; ++, [IC.sub.50] 1-[micro]M; +,
[IC.sub.50] 10-100 [micro]M -, [IC.sub.50] > 100 [micro]M; ND
not determined

Table 3

Summary of the isoquinoline alkaloids in each inhibition potency
category.

Category            CYP2D6n =  CYP3A4n =  CYP2C19n  CYPlA2n=36(%)
([IC.sub.50]        34(%)      36(%)      = 36(%)
value)

Potent              21(61.8)   15(41.7)   3(8.3)    1(2.8)
(< 1 [micro]M)

Moderate (1-10      5(14.7)    15(41.7)   12(33.3)  10(27.8)
[mu]M)

Weak (> 10[mu]M)    7(20.6)    4(11.1)    16(44.4)  23(63.9)

Non-inhibitor (>    1 (2.9)    2(5.5)     5(13.9)   2(5.5)
100[micro]M)

Category            CYP2C9n =  CYP2B6n =  CYP2C8n=36(%)  CYP2A6n=36(%)
([IC.sub.50]        36(%)      36 (%)
value)

Potent (< 1 [mu]M)  0(0)       0(0)       0(0)           0(0)

Moderate (1-10      6(16.7)    4(11.1)    3(8.3)         0(0)
[mu]M)

Weak (> 10[mu]M)    14(38.9)   19(52.8)   17(47.2)       1(2.8)

Non-inhibitor (>    16(44.4)   13(36.1)   16(44.4)       35(97.2)
100[micro]M)

The CYP forms most potently inhibited are presented first.


CYP2D6 was inhibited most potently by the protopine alkaloids (6/8 potently) and protoberberine alkaloids (14/20 potently) as well as the phthalideisoquinoline alkaloide capnoidine. The aporphine alkaloids and the spirobenzylisoquinoline alkaloide, parfumine, inhibited CYP2D6 only slightly (2/6 aporphine alkaloids with moderate potency). In contrast to CYP2D6, CYP3A4 was inhibited by the aporphine alkaloids. Protopine and protoberberine alkaloids were more potent inhibitors of CYP2D6 than CYP3A4. Alkaloids inhibiting CYP2C19 belonged mostly to the protopine and protoberberine alkaloids.

Fig. 1 illustrates the inhibition of CYP1A2, CYP2A6, CYP2D6, and CYP3A4 by apomorphine (7). Apomorphine was a moderately potent inhibitor of CYP1A2 with an [IC.sub.50] value of 4.8 [+ or -] 1.8 (< 1 [micro]M)M (mean [+ or -] SEM), and a weak inhibitor of CYP2D6 and CYP3A4 with [IC.sub.50] values of 11.3 [+ or -] 5.5 (< 1 [micro]M)M and 34.0 [+ or -] 8.1 (< 1 [micro]M)M, respectively. In contrast, apomorphine was not able to inhibit CYP2A6 ([IC.sub.50] > 100 (< 1 [micro]M)M).

Discussion

The results of this study provide an extensive perspective of the interaction of novel plant isoquinoline alkaloids with the most important human CYP enzymes. Overall, there were clear differences in the ability of these alkaloids to inhibit individual CYP forms. CYP2D6 and CYP3A4 were most potently inhibited, whereas CYP1A2, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 were inhibited to a lesser degree. One notable feature was the inability of the alkaloids to affect CYP2A6. This is consistent with the known small and restricted active site of this CYP form (Yano et al. 2006). Several new potent inhibitors against CYP3A4, CYP2D6, CYP2C19, and CYP1A2 were found. Of particular interest is the selective inhibition of CYP3A4 by corydine (11), parfumine (13) and 8-methyl-2,3,10,11-tetraethoxyberbine (31).

Fluorometric high-throughput inhibition assays of CYP enzymes are widely used for drug interaction screening particularly during the preclinical drug discovery stages. Fluorometric inhibition screening has been found to be comparable with other potential screening procedures employed, such as HPLC and mass spectrometry detection (Turpeinen et al. 2006; Pelkonen et al. 2008; Kapitulnik et al. 2009). When many compounds of unknown and varying inhibitory potency are being screened, the [IC.sub.50] value is a practical readout of the relative effects on CYP enzyme activity under well-controlled conditions. By using suitable in vitro probes and careful selection of interacting drugs during in vivo studies, the potential for drug interactions can be evaluated early in the development process (Huang et al. 2007). Therefore a fluorometric high-throughput inhibition assay was used in this study to determine interaction potency between CYPs and plant isoquinoline alkaloids.

CYP3A4 has a pivotal role in xenobiotic metabolism, and it has been estimated to be involved in the metabolism of up to 50% of all drugs in clinical use. The active site of CYP3A4 is very large and flexible allowing the binding and subsequent metabolism of structurally very diverse compounds. The substrate binding is principally based on hydrophobicity with some steric interactions (Williams et al. 2004; Yano et al. 2004; Zhou 2008). In this study, 42% of the tested isoquinoline alkaloids were potent and 42% moderate inhibitors of CYP3A4. The current findings will add to current understanding of the structure-activity relationship of ligands at the active site of CYP3A4. Corydine (11), parfumine (13), and 8-methyl-2,3,10,11-tetraethoxyberbine (31) are novel CYP3A4-selective inhibitory compounds. They inhibited potently CYP3A4 and weakly or not at all the other CYP forms. Therefore they may be used as selective inhibitors of CYP3A4 to study CYP mediated metabolism of test compounds in vitro.

The typical CYP2D6 substrates are lipophilic bases with a planar hydrophobic aromatic ring and a nitrogen atom which can be protonated at physiological pH (Lewis 2004). The tested isoquinoline alkaloids possess these properties. Lipophilicity and amine basicity are considered to be the two critical determinants of substrate binding to CYP2D6. A number of CYP2D6 substrates and other compounds have been found to inhibit CYP2D6 (Lewis 2004; Want et al. 2009). In this study, as high as 62% of the alkaloids were potent inhibitors of CYP2D6. However, none of them was as selective as the three found inhibitors of CYP3A4. However, cryptopine (3), allocryptopine (4), scoulerine (26), mecambridine (27), 2,3,10,11-tetramethoxyberbine (28), 8-ethyl-2,3,10,11-tetramethoxyberbine (29) and 2,3-dimethoxyberbine (36) had some selectivity towards CYP2D6, and these compounds add to the list of known CYP2D6 ligands (Lewis 2004; Wang et al. 2009).

[FIGURE 1 OMITTED]

The diversity of the biosynthetic pathways in plants has provided a variety of lead structures that have been used in drug development and which have been estimated to account for more than 50% of current drugs. The plant kingdom still remains a treasure trove of new molecules with therapeutic potantial (Stevigny et al. 2005; Newman and Cragg 2007). Alkaloids exist in a number of herbal medicines as their major biologically active constituents. Herbal alkaloids may be substrates, inducers, or inhibitors of various CYPs. For example, some vinca alkaloids (e.g. vinblastine) are metabolized by CYP3A4, and this has been associated with tumour resistance (Yao et al. 2000; Zhou et a. 2003).

Apomorphine was the only compound in this study currently used as a drug. It is administered subcutaneously to treat Parkinson's disease. Despite being a derivative of the opioid, morphine, apomorphine lacks the narcotic properties and other opiate effects of its parent compound. Apomorphine is not commercially available for oral administration because of its extensive inactivation via hepatic first-pass metabolism and poor bioavailability. The route of metabolism after subcutaneous administration of apomorphine in humans is not known but in several animal species, apomorphine undergoes metabolism by glucuronidation and O-methylation (Chen and Obering 2005). In this study, apomorphine did not possess any significant inhibitory capabilities against human CYPs (moderate potency towards CYP1A2, CYP2C8, and CYP2C9, and weak potency towards CYP2B6, CYP2C19, CYP2D6 and CYP3A4). For CYP1A2, CYP2D6, and CYP3A4, these results are consistent with those obtained with human hepatic microsomes in vitro (Argiolas and Hedlund 2001).

The prodrug codeine is extensively metabolized by CYP2D6, and the clinical analgesic effect of codeine is mainly attributed to its conversion to morphine (Lotsch et al. 2009). Like apomorphine, also buprenorphine (a semisynthetic opioid derived from thebaine, naturally occurring alkaloid of Papaver somniferum, and used as substitution therapy in the treatment of opioid dependence), undergoes extensive first-pass metabolism and therefore has very low oral bioavailability (Elkader and Sproule 2005). In addition, it is known that buprenorphine is extensively metabolized by N-dealkylation primarily via CYP3A4 (Elkader and Sproule 2005).

To learn whether the current alkaloids are present in TCM, a search of a commercial TCM Database (NiceData Software 2005) was carried out. This database includes 10,458 individual compounds isolated from TCM plants, 4636 TCM medicinal plants, and their therapeutic utilities. Of the present 36 alkaloids, 19 were found in the database. For 10 alkaloids (1, 3, 4, 9, 10, 11, 14, 17, 27, and 28) one or more TCM pharmacological or therapeutic effects were listed. Three plants listed in the database (Papaver somniferum L., Chelidonium majus L., and Corydalis cava L.) were related to alkaloids 1, 3, 4, 9, 10, 17, and 22. In contrast to TCM, isoquinoline-containing plants are currently not commonly used in European medicine, as exemplified by the fact that the European Pharmacopoeia (Ph. Eur.) includes only Fumaria officinalis L.

In conclusion, this study presents a detailed account on the ability of a series of structurally related alkaloids to interact with all major human metabolizing CYP enzymes. To date, here is only a sparse literature on interactions between plant alkaloids and human CYP enzymes. However, there is information about the preferred substrates and the functionality of the major human CYP enzymes and these present results agree with these general principles. The current study indicated high-affinity interaction of several isoquinoline alkaloids to several CYP enzymes. Future experiments on the actual metabolic turnover of the compounds will yield insight into their possible metabolic fate in humans. This dataset also provides an opportunity to carry out detailed structure-activity relationship studies. Several novel compounds with selectivities towards individual CYP forms were identified and these have the potential to be developed as form-specific inhibitors for further in vitro CYP screenings.

Conflicts of interest

The authors have no conflicts to disclose.

Acknowledgements

We thank Hannele Jaatinen from the University of Eastern Finland for technical help, Jiri Slavik and Jiri Dostal from the Masaryk University of Brno, Czech Republic for the samples of hunnemannine, allocryptopine and mecambridine, Dr. Tuomo Laitinen, from the University of Eastern Finland for help with TCM databases, and Dr. Ewen MacDonald from the University of Eastern Finland for help in preparing the manuscript. Funding for this research was received from the Finnish Graduate School of Toxicology. The historical samples (the invaluable Gadamer collection) were kindly provided by the Philipps University of Marburg, Germany.

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Kaisa A. Salminen (a), *, Achim Meyer (b), Lenka Jerabkova (a), Laura E. Korhonen (a), Minna Rahnasto (a), Risto O. Juvonen (a), Peter Imming (b), Hannu Raunio (a)

(a) School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland

(b) Institut fur Pharmazie, Abteilung Pharmazeutische Chemie, Martin-Luther-Universitat Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany

* Corresponding author. Tel.: +358 40 355 3774; fax: +358 17 162424. E-mail address: kaisa.salminen@uef.fi (K.A. Salminen).

0944-7113/$ - see front matter [C] 2010 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2010.08.012
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Author:Salminen, Kaisa A.; Meyer, Achim; Jerabkova, Lenka; Korhonen, Laura E.; Rahnasto, Minna; Juvonen, Ri
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Apr 15, 2011
Words:4961
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