scientiam dulce hauriens
Dr. Adrian Moore
Department of Pharmacy, Health and Well-being
Faculty of Applied Sciences
UoS Sciences Complex
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>£20 million invested in capital development programmes
– new-build projects
– refurbishment of existing estate
– on-going developments
To provide modern, well-equipped facilities to support high quality teaching and
learning, real-life applied research, and support for business and our wider community
– One-North East
– EU ERDF (innovation, enterprise and business support)
Health and Science Academy
“provide the best postgraduate teaching and research facilities to accommodate the
professional development needs of the pharmaceutical supply chain and support
small companies and business start-up ventures in the region”
Computational Methods
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Comprehensive investigation of molecules, their reactions and interactions
– chemical structures / biological reactions at the molecular level
– discovery of new drugs that target particular cells
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Pharmacophore or Quantitative Structure Activity Relationship (QSAR)
– rationalise existing biological data to make informed choices as to the next
series of compounds to make
Rational Drug Design
– good quality structural information is available of intended biological target
enzyme, receptor protein or strand of DNA/RNA
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Molecular mechanics (MM) methods
Quantum mechanical (QM) methods
– highly computationally resource intensive
– UoS cluster computer application framework
Synthesis
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Traditional; research -> medium scale
– Range of current projects
Flow chemistry
– wide range of reactions possible
– production of libraries or on multi-gram scale
– safely use highly reactive/hazardous reagents
– easily uses solid-phase reagents
– good reproducibility -> reduced scale-up
issues
– inherent reaction control and selectivity
– wide temperature range -> superheating
gives faster reactions
– reduced scale limitations -> quick reaction
evaluation
O
O
pyBOP, TEA
OH
X
+
H2 N
Y
O
X
N
H
X
N
Y
N
N
O
O
OH
N
N
X
N
H
Y
N
O
N
H
X
Y
OH
N
N
N
20 compound library, synthesis and aqueous work-up, high yield and high purity (LCMS), no purification
40 mg of each compound synthesised, mass recovery 87.5 % ± 1.5
SPR – Linked to Flow Synthesis
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Analyse library molecular substrate/target interactions in real time
– proteins
– nucleic acids
– lipids and membrane-associated molecules
– carbohydrates
– whole cells
– viruses/bacteria
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Obtain a wide range of critical, binding-related data
– specificity
– binding partners
– affinity
– kinetics
– concentration
– thermodynamics
Chip:
• hydrophilic
• flexible
• low non-specific binding
• high binding capacity
• easy to activate and use for covalent attachment of ligand
• withstands extensive regeneration
Attachment of ligand
• amine coupling
• ligand thiol coupling
• surface thiol coupling
• maleimide coupling
• aldehyde coupling
Analyte
Ligand
buffer
sample
buffer
association
dissociation
Separation Science:
Areas of Expertise
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All areas of pharmaceutical and biomedical analysis
– medicinal plant extracts
– biomarker analysis
– cleaning validation
– drug bioanalysis
– preparative isolation of API
– preparative isolation of related substances
– rapid API screens / related substances
– chiral screening
– stability screening
– confirmation of structure
– unknown identification
Analytical Science: Capability
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HPLC / UHPLC (better efficiency, high throughput)
• ELSD (non volatile, nonchromaphoric compounds)
• RI (more volatile, nonchromaphoric compounds)
• fluorescence
• diode array
• single quad MS cooled auto samplers on MS (to 4C)
• UHD-QTOF (high resolution and mass accuracy)
• prep-LC
GC-FID, GC-Q, GC-QQQ
• SPME, head space, and cold-on-column
MALDI-TOF
CE, CE-MS
• proteins and complex/biological analysis especially
atomic absorption, flame photometry, UV, IR,
fluorometry, luminometry, Karl Fischer, ionchromatography, logD, pKa, TGA-DSC, microscopy (SEM,
TEM, confocal)
Separation Science:
Rapid LC Related Substances Method
UPLC of paroxetine and all related substances in 1.2 minutes
BEH C18 (1.7 mm) (50 mm x 2.1 mm ID); 1 mg ml-1 paroxetine and related substances at ~ 0.002 mg ml-1.
UV detection - 295 nm. Mobile phase component A (water – THF – TFA (90:10:0.5, v/v/v)), B
(acetonitrile – THF – TFA (90:10:0.5, v/v/v)). Resolution was maintained when using a steep gradient
profile throughout and also when the temperature was raised to 80 oC.
Complex
Samples
Unknown identification
tmsi glu
, 11-Dec-2009 +
tmsiglu 561 (5.457) Rf (7,5.000)
73.0274
573980672
100
140.1913
415662080
98.0790
338937856
125.0983
301457408
%
70.0108
58.9439 61574144
31477320
74.0328
50367488 95.0674
7497911
99.0827
38347776
59
69
79
89
99
109
119
141.1304
58639360
126.1006
35997696
73
100
0
49
112.1263
7868672
129
213.1751
2671328
139
149
159
169
179
189
199
209
219
265.0
6003
229
249
259
73
100
98
140
98
125
50
140
Si
50
N
125
0
239
55 62
50
60
70
70
84
80
109
90
100
110
213
120
C:\ TurboMass\ Default.pro\ Data\ tmsiglu: Scan 561 (0. min)
130
140
150
160
170
180
190
200
210
Side by Side MF=944 RMF=968
265
220
230
240
250
260
N
270
1H-Imidazole, 1-(trimethylsilyl)-
70
55
0
50
84
63
60
70
80
112
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
Complex Samples – Principle Analysis
• Metabolic profiling of low MW components in biological fluid samples
• Pathological conditions can create metabolic disruptions detectable in the metabolite
content of biofluids
• variation in concentration and relative proportion
• Identify affected pathway -> novel approach to treatment
NMR Services
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Structure confirmation / determination
– spectra with or without interpretation
– cooled auto-sampler for biological/unstable samples
– fast turnaround routine service
Impurity profiling / identification by NMR / MS
Characterisation of peaks in liquid chromatograms, LC-NMR
– characterisation of unstable components in complex mixtures
– structure elucidation of minor components in complex
mixtures
– detailed structural information where LC-MS is inappropriate
Identification of Natural Products :
Comparison Extracted Product/Standard
Extract
High resolution MS :
M = 610 g mol-1, C27H30O16
1-D NMR 500 MHz LC-1H NMR
spectrum of approx. 2 mg of rutin,
isolated from extracts of Sophora
japonica , in D2O-CH3CN
[solvent suppression at d1.90 (CH3CN)
and d4.46 (residual water)]
Standard
S 24795
S 24795 completed Phase I Clinical Studies
Synthesis developed to semi-production scale (500 kg)
negative allosteric modulator at nicotinic receptors
wide-spectrum pro-cognitive, psycho-behavioural activity
Electrophysiology
a7 : 42 ± 2 mM
a4b2 : 230 ± 16 mM
(rat – expressed on Xenopus Laevis oocytes)
Binding studies
a7 > 10000 nM, a4b2 > 10000 nM
(a1)2bdg ~ 10000 nM, a3b4 > 10000 nM
security binding (80 sites) – no negative interactions
CaCo2 100% hCMEC/D3 90%
Microsomes rat 31%, human 75%
in vitro clastogenotoxicity!
(minor metabolite related, < 0.2% profile)
Identification of Metabolite:
Extraction From Rat Bile
Parent compound
Aromatic CH
NH
Metabolite ; 352 scans (20min)
2 µg
MS analysis : +16 → oxidation
MS/MS → oxidation on one of the 3 substituted aromatic rings
exact position of hydroxylation from LC-NMR
WEEK
1
Pharmaceutics
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Pre-formulation studies
– phase transitions / microscopic changes
– polymorphic form studies
– solubility screening
– pKa, log P/D determination
– solution and solid-state stability, degradation studies, degradation product identity
– excipient and active compatibility studies
– particle size measurements
– powder electrostatics
– physical characterisation of drug delivery systems and vehicle optimisation
– problem solving of existing processes and formulations
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Formulation
– tablet, capsule, solution, suspension, emulsion, injectable, patch etc.
– protein stabilisation and delivery
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Aseptic capability – category 2 clean room
Dr. Adrian Moore
Department of Pharmacy, Health and Well-being
Faculty of Applied Sciences
University of Sunderland
Dale Building, Room 1.03
Sciences Complex
Wharncliffe Street
Sunderland
SR1 3SD
T : +44 (0)191 515 2554
F : +44 (0)191 515 3405
E : adrian.moore@sunderland.ac.uk