Clinically-approved drugs against CNS diseases as potential

Oct 15, 2018 - ... feasible due to advantages such as; permeability of the blood-brain barrier; established pharmacokinetics and dynamics; FDA approva...
0 downloads 0 Views 3MB Size
Subscriber access provided by Gothenburg University Library

Article

Clinically-approved drugs against CNS diseases as potential therapeutic agents to target brain-eating amoebae Ayaz Anwar, Kavitha Rajendran, Ruqaiyyah Siddiqui, Muhammad Raza Shah, and Naveed Ahmed Khan ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00484 • Publication Date (Web): 15 Oct 2018 Downloaded from http://pubs.acs.org on October 17, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

ACS Chemical Neuroscience

Clinically-approved drugs against CNS diseases as potential therapeutic agents to target brain-eating amoebae

3 4

Ayaz Anwar1*, Kavitha Rajendran1, Ruqaiyyah Siddiqui1, Muhammad Raza Shah2, Naveed

5

Ahmed Khan1

6 7

1: Department of Biological Sciences, School of Science and Technology, Sunway University,

8

Malaysia. 2: HEJ Research Institute of Chemistry, International Center for Chemical and

9

Biological Sciences, University of Karachi, Karachi, Pakistan.

10

[email protected]; [email protected]; [email protected];

11

[email protected]; [email protected]

12 13

Short title: CNS drugs against brain-eating amoebae

14 15 16

*Corresponding address: Department of Biological Sciences, School of Science and Technology,

17

Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-8622. Ext: 7169. Fax: 60-

18

(0)3-5635-8630. E-mail: [email protected]; [email protected]

1 ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

20 21

Page 2 of 31

Abstract Central nervous system (CNS) infections caused by free-living amoebae such as

22

Acanthamoeba species, Naegleria fowleri etc. are rare but fatal. A major challenge in the

23

treatment against the infections caused by these amoebae is the discovery of novel compounds

24

that can effectively cross the blood-brain barrier to penetrate CNS. It is logical to test clinically-

25

approved drugs against CNS diseases for their potential antiamoebic effects since they are

26

known for effective blood-brain barrier penetration and effect eukaryotic cell targets. The

27

antiamoebic effects of clinically available drugs for seizures targeting gamma-amino butyric acid

28

(GABA) receptor and ion channels were tested against Acanthamoeba castellanii (A. castellanii)

29

belonging to the T4 genotype and Naegleria fowleri (N. fowleri). Three such drugs namely;

30

Diazepam (Valium), Phenobarbitone (Luminal), Phenytoin (Dilantin) and their silver

31

nanoparticles (AgNPs) were evaluated against both trophozoites and cysts stage. Drugs alone

32

and drugs conjugated silver nanoparticles were tested for amoebicidal, cysticidal and host-cells

33

cytotoxicity assays. In vitro amoebicidal assay showed potent amoebicidal effects for Diazepam,

34

Phenobarbitone, and Phenytoin-conjugated AgNPs as compared to drugs alone against A.

35

castellanii and N. fowleri. Nanoparticles were synthesized by sodium borohydride reduction of

36

silver nitrate with drugs as capping agents. Drugs conjugated nanoconjugates were characterized

37

by ultraviolet-visible (UV-vis), and Fourier transform infrared (FT-IR) spectroscopies, and

38

atomic force microscopy (AFM). Furthermore, both drugs and drugs conjugated AgNPs showed

39

compelling cysticidal effects. Drugs conjugations with silver nanoparticles enhanced their

40

antiacanthamoebic activity. Interestingly, amoeba-mediated host cells cytotoxicity was also

41

significantly reduced by drugs alone as well as their nanoconjugates. Since, these drugs are being

42

used to target CNS diseases, their evaluation against brain-eating amoebae seems feasible due to

43

advantages such as; permeability of the blood-brain barrier; established pharmacokinetics and 2 ACS Paragon Plus Environment

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

44

dynamics; FDA approval etc. Given the limited availability of effective drugs against A.

45

castellanii, the clinically available drugs tested here present potential for further in vivo studies.

46

Keywords: Acanthamoeba; Naegleria; Diazepam (Valium); Phenobarbitone (Luminal);

47

Phenytoin (Dilantin); Nanoparticles.

48

Introduction

49

Infectious diseases involving CNS due to brain-eating amoebae; Acanthamoeba species,

50

Balamuthia mandrillaris, and Naegleria fowleri are rare but almost always result is mortality.1

51

Acanthamoeba castellanii (A. castellanii) is a facultative parasite and most frequently causes a

52

painful, sight intimidating infection, Acanthamoeba keratitis, and devastating granulomatous

53

amoebic encephalitis.2 Acanthamoeba keratitis most commonly affects contact lens wearers.3 A.

54

castellanii life cycle consists of two forms; reproductive trophozoite, and sturdy cysts.2 Whereas,

55

Naegleria fowleri causes deadly primary amoebic encephalitis (PAM).4,5 The current

56

management and treatment of the brain-eating amoebae infections include combination of

57

multiple drugs e.g., chlorhexidine, polyhexamethylene biguanides and propamidine with

58

ketoconazole, neomycin, amphotericin B, milfetosine, sulfadiazine etc. There are some

59

limitations to the use of these drugs including; resistance of cysts to uptake the drugs hence risk

60

of recurrence, host-cells cytotoxicity due to prolonged treatment, etc.6 Albeit challenges in the

61

discovery of novel drugs and approvals for clinical use, re-purposing of clinically-approved

62

drugs against new diseases targets is a useful strategy. The discovery of novel compounds which

63

can penetrate blood-brain barrier and target amoebae residing in CNS is indispensable, hence, to

64

test the clinically-approved drugs for CNS diseases against brain-eating amoebae has a practical

65

rationale.

3 ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

66

Page 4 of 31

Nanoparticles are effective drug delivery vehicles associated with increasing the

67

bioavailability of their cargoes.7 Their small size ensures specific target transportation and

68

minimal side effects at lower concentration.7 Silver nanoparticles (AgNPs) are anticipated as

69

next generation class of antimicrobials due to their known affinity towards DNA of pathogens

70

and production of reactive oxygen species.8 There are several reports on antimicrobial activities

71

of AgNPs in particular against bacteria.9 Currently, AgNPs uses in the topical burn ointments,

72

wound healing materials for dressings in injuries, and their impregnation in medical devices such

73

as catheters, grafts etc., are subject of vast interest.10-12 However, their utility against protists

74

infectious diseases is limited. AgNPs are recently developed as viable alternatives in treatment

75

against brain-eating amoebae, A. castellanii and N. fowleri.13-15

76

Herein we demonstrated the effects of clinically available drugs; Diazepam,

77

Phenobarbitone, and Phenytoin on A. castellanii and N. fowleri. Diazepam is clinically used to

78

treat anxiety disorder, and sometimes used with other drugs to control seizures.16 The oral dosage

79

of Diazepam ranges from 2-10 mg 2-3 times a day depending on severity of disease.17

80

Phenobarbitone is an anticonvulsant drug which is mainly used to control seizures in epileptic

81

and bipolar patients.18 The recommended dosage of Phenobarbitone is 15-18 mg/kg via

82

intravenous administration for epileptic patients.19 Phenytoin is another

83

anticonvulsant/antiepileptic drug taken orally in the form of capsule with the dosage of 30 mg

84

once daily.20 All selected drugs are FDA approved, and are in the WHO list of essential

85

medicines needed in health system. Besides using these drugs alone, we also tested whether the

86

conjugation of silver nanoparticles with these drugs enhanced their bioactivities against brain-

87

eating amoebae. Testing these CNS targeting drugs and their silver nanoconjugates against A.

88

castellanii and N. fowleri resulted in antiamoebic therapy. All of drugs and nanoparticles showed 4 ACS Paragon Plus Environment

Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

89

remarkable amoebicidal and cysticidal properties and reduced amoeba-mediated host cells

90

cytotoxicity.

91

Results and discussion

92

CNS infectious diseases caused by brain-eating amoebae such as Granulomatous amoebic

93

encephalitis (GAE), Primary amoebic encephalitis (PAM) etc. are rare, and due to lack of

94

knowledge difficult to diagnose as well as no single available effective therapeutic option make

95

them lethal in almost all cases.21,22 GAE due to A. castellanii is a fatal infection of brain and

96

spinal cord generally affecting the immunocompromised patients.23 GAE symptoms typically

97

progress over several weeks to months. Once the CNS is affected, the symptoms may include;

98

headaches and fever, stiff neck, nausea, tiredness, lack of focus and body coordination, seizures,

99

etc.1 PAM due to N. fowleri is a severe, fulminating infection of the CNS.5 The therapeutic

100

agents used to treat and manage infections caused by brain-eating amoebae include

101

chlorhexidine, amphotericin B, voriconazole, rifampicin, pentamidine, polyhexamethylene

102

biguanide, azithromycin, miltefosine and/or a mixture of these drugs, but they have limitations

103

due to their reduced bioavailability and minimum inhibitory concentration (MIC) at the site of

104

infection, off-site cytotoxicity and limited potency against cysts.24 Therefore, there is an urgent

105

need to find novel approaches for development of new and effective therapeutic alternatives, as

106

well as administration strategies.

107

Herein, we selected three drugs clinically used against CNS diseases including

108

Diazepam, Phenobarbitone, and Phenytoin (benzodiazepine, barbiturate, and hydantoin

109

respectively) to evaluate their activity against brain-eating amoebae. Their brief mode of action

110

in humans is summarized in Table 1. Benzodiazepines bind to benzodiazepine receptors resulting

111

in relaxation of muscles, and cause calming effects which effects motor coordination and 5 ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 31

112

memory, and show anticonvulsant activity.25 Benzodiazepine receptors are known to be coupled

113

to gamma-aminobutyric acid-A (GABAA) receptors, which effects GABA by increasing its

114

affinity for the receptor. 25 This binding causes opening of the chloride ions channel, resulting in

115

a hyperpolarization on the cell membrane which control seizures and leads to stabilization.25

116

Phenytoin acts on sodium ion channels at the neuronal cell membrane, and limits spreading of

117

seizure activity. As a result of promoting sodium efflux from neurons, it controls the threshold

118

against excitability caused by excessive stimulation capable of reducing gradient of sodium on

119

the membrane.26 Phenobarbitone is another promoter of GABAA receptors, which has the

120

potential of increasingseizure threshold and reducing the spread of seizure. It is also known to

121

inhibit calcium channels, which aids in a decrease in excitatory transmitter release.27 Diazepam

122

is known to penetrate blood-brain barrier much faster than Phenobarbitone and Dialantin.28 A.

123

castellanii is known to have specific binding sites with benzodiazepines in mitochondrial

124

membrane with proteins similar as mammalian cells called mitochondria benzodiazepine

125

receptors (MBR).29 Thus, the alteration in mitochondrial activity by proteins-Diazepam

126

interactions with amoebae may be the reason for such a huge reduction in its viability. Moreover,

127

MBR are also associated with several other functions such as; importance in steroids

128

biosynthesis, control of mitochondrial respiration, cells proliferation, transfer of calcium ions

129

etc.29 Hence the mode of action of effects of Diazepam against brain-eating amoebae is proposed

130

to be a multifactor action of above features. Phenobarbitone and Phenytoin are effective

131

sodium/calcium channels inhibitors which play pivotal role in the metabolism functioning of

132

brain-eating amoebae and their genotype modification30,31 which may be responsible for

133

inhibition of these processes. Since the usage of anticonvulsant drugs such as those used in this

134

study is common in amoebic encephalitis management, we anticipate that these findings provide

6 ACS Paragon Plus Environment

Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

135

a novel strategy to develop the potent amoebicidal agents combined with their known antiseizure

136

effects which can be used to minimize the suffering of patients. Based on above discussion, it

137

was logical to test the antiamoebic activity of these drugs and their nanoconjugates.

138

Characterization of Diazepam, Phenobarbitone, and Phenytoin conjugated Silver

139

nanoparticles via UV-visible spectrophotometry, AFM, and FT-IR spectroscopy.

140

Drugs conjugated AgNPs were analyzed by UV-vis, FT-IR spectroscopy, and AFM for

141

characterization. Diazepam conjugated silver nanoparticles (DZP-AgNPs), Phenobarbitone

142

conjugated silver nanoparticles (PBT-AgNPs), and Phenytoin conjugated silver nanoparticles

143

(PTN-AgNPs) were successfully stabilized by these drugs. UV-vis spectra of DZP-AgNPs, PBT-

144

AgNPs, and PTN-AgNPs showed a representative surface plasmon resonance (SPR) band for

145

AgNPs at 415, 420 and 428 nm respectively (Fig. 1a), which suggest the formation of

146

nanoconjugates. AFM analysis was performed for determination of size and morphology of

147

AgNPs conjugated with drugs. All nanconjugates of drugs were found to be spherical with a

148

wide size distribution (10-200 nm) due to rapid reduction by using sodium borohydride (Fig. 1b-

149

d). The comparative FT-IR spectral analysis of Phenytoin sodium with the PTN-AgNPs was

150

carried out to identify the stabilizing functional groups in drug as a representative example. The

151

FTIR spectrum of phenytoin sodium showed the characteristic signals of functional groups

152

present in PTN. The peak at 3318 cm-1 was due to N-H stretch while peak at 1397 cm-1 was due

153

to N-H bending. The peaks at 3066, 1592 and 1492 cm-1 were due to aromatic ring stretching

154

vibration. The C=O of amide group appeared as a split peaks at 1674 and 1687 cm-1.32 After the

155

formation of PTN-AgNPs, the N-H stretching shifted from 3318 to 3449, whereas the N-H

156

bending signal was observed with slight red shift at 1383 cm-1. These results clearly indicate the

157

involvement of N-H functionality present in the hydantoin ring in the stabilization of AgNPs. 7 ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 31

158

Silver nanoparticles conjugation with Diazepam, Phenobarbitone, and Phenytoin significantly

159

enhanced their amoebicidal effects against A. castellanii

160

Amoebicidal assays were carried out at various concentrations, the result (Fig. 2)

161

revealed that CNS drugs conjugated with AgNPs exhibited significant amoebicidal potency

162

against A. castellanii as compared to CNS drugs when used alone (P < 0.05, two-sample T test

163

and two-tailed distribution). On the other hand, neither AgNPs alone nor DMSO used as solvent

164

control showed any effects. All drugs nanoconjugates inhibited the viability of A. castellanii to

165

104 as compared to controls and initial inoculum i.e., 105.

166

Diazepam, Phenobarbitone, and Phenytoin alone and their nanoconjugates both inhibited

167

encystation of A. castellanii

168

The potential of conjugation of AgNPs with Diazepam, Phenobarbitone, and Phenytoin

169

on encystment of A. castellanii was evaluated to establish the contrasting encystation

170

differentiation as compared to drugs alone. The encystation media induced formation of cysts

171

and 4.13x104 number of cysts were enumerated without any drugs (Fig. 3). However, the

172

addition of drugs and their nanoconjugates, resulted in significant reduction in encystation of

173

trophozoites (P < 0.05, two-sample T test and two-tailed distribution). The difference between

174

encystation inhibition capabilities can be observed only in the case of Diazepam and

175

Phenobarbitone with respect to their nanoconjugates with latter being more effective, whereas

176

Phenytoin and PTN-AgNPs both produced same effects.

177

Diazepam, Phenobarbitone, Phenytoin, and their AgNPs conjugates inhibited excystation

178 179

To test the cysticidal effects of drugs nanoconjugates, cysts were prepared as described in materials and methods section and were incubated with various concentrations of drugs and 8 ACS Paragon Plus Environment

Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

180

drugs coated nanoconjugates in growth medium PYG. In the absence of CNS drugs, A.

181

castellanii cysts emerged as viable trophozoites (Fig. 4). Contrary, both drugs and their AgNPs

182

showed significant inhibition in excystation of A. castellanii cysts (P < 0.05, two-sample T test

183

and two-tailed distribution). However, the enhanced effects of nanoparticles conjugation on

184

drugs were clearly evident at 10 µM concentration. On the other hand, AgNPs alone had limited

185

effects on excystation.

186

CNS drugs and their nanoconjugates reduced host cells cytopathogenicity of A. castellanii

187

A. castellanii mediated host cells cytotoxicity was also evaluated by cytopathogenicity

188

assays using LDH determination as a measure of cells damage. Triton X-100 treated cell

189

cytotoxicity was considered as 100% while all other cytotoxicity measured by LDH assay were

190

plotted comparatively to this positive control (Fig. 5). A. castellanii alone produced more than

191

80% host cell cytotoxicity, whereas Chlorhexidine pretreated amoeba showed no damage to

192

cells. Instead, A. castellanii pretreated with drugs alone and drugs conjugated AgNPs caused

193

significant reduction in host cell cytotoxicity at 10 µM and showed minimal cell damage (P