Dr. Adrian MooreDepartment of Pharmacy, Health and Well-being

Faculty of Applied Sciences

scientiam dulce hauriens

UoS Sciences Complex• >£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• Comprehensive investigation of molecules, their reactions and interactions

– chemical structures / biological reactions at the molecular level – discovery of new drugs that target particular cells

• 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

• Molecular mechanics (MM) methods• Quantum mechanical (QM) methods

– highly computationally resource intensive– UoS cluster computer application framework

Synthesis• 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

XO

ON

NN

NH

O

YX

N

NN

OH

N

NN

OH

NH

O

YX

NH

O

YXNH2

YX

O

OH+

pyBOP, TEA

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 • Analyse library molecular substrate/target interactions in real time

– proteins– nucleic acids– lipids and membrane-associated molecules – carbohydrates– whole cells– viruses/bacteria

• Obtain a wide range of critical, binding-related data– specificity – binding partners – affinity– kinetics – concentration– thermodynamics

Analyte

Ligand

Attachment of ligand• amine coupling • ligand thiol coupling• surface thiol coupling• maleimide coupling• aldehyde coupling

Chip:• hydrophilic• flexible• low non-specific binding• high binding capacity• easy to activate and use for covalent attachment of ligand• withstands extensive regeneration

buffer

association

buffer

dissociation

sample

Separation Science:Areas of Expertise

• 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• 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, ion-chromatography, 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

5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 00

5 0

1 0 0

5 5 6 27 0

7 3

8 4

9 8

1 0 9

1 2 5

1 4 0

2 1 3 2 6 5

5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 00

5 0

1 0 0

5 56 3

7 0

7 3

8 4

9 8

1 1 2

1 2 5

1 4 0 S i

N

N

49 59 69 79 89 99 109 119 129 139 149 159 169 179 189 199 209 219 229 239 249 259 269m/z0

100

%

tmsiglu 561 (5.457) Rf (7,5.000) Scan EI+ 5.74e873.0274

573980672

70.01086157414458.9439

31477320

140.1913415662080

98.0790338937856

74.032850367488 95.0674

7497911

125.0983301457408

99.082738347776 112.1263

7868672

126.100635997696

141.130458639360

213.17512671328

265.0959600379

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• 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

Extract

Standard

Identification of Natural Products :Comparison Extracted Product/Standard

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)]

S 24795 completed Phase I Clinical StudiesSynthesis developed to semi-production scale (500 kg)

negative allosteric modulator at nicotinic receptorswide-spectrum pro-cognitive, psycho-behavioural activity

Electrophysiology

a7 : 42 ± 2 mM a4b2 : 230 ± 16 mM(rat – expressed on Xenopus Laevis oocytes)

Binding studiesa7 > 10000 nM, a4b2 > 10000 nM

(a1)2bdg ~ 10000 nM, a3b4 > 10000 nMsecurity 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)

S 24795

Identification of Metabolite:Extraction From Rat Bile

MS analysis : +16 → oxidation

MS/MS → oxidation on one of the 3 substituted aromatic rings

exact position of hydroxylation from LC-NMR

Aromatic CH NH

Metabolite ; 352 scans (20min)

Parent compound

2 µg

Pharmaceutics• 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

• Formulation– tablet, capsule, solution, suspension, emulsion, injectable, patch etc.– protein stabilisation and delivery

• Aseptic capability – category 2 clean room

WEEK

1

Dr. Adrian MooreDepartment of Pharmacy, Health and Well-being

Faculty of Applied SciencesUniversity of SunderlandDale Building, Room 1.03

Sciences ComplexWharncliffe Street

SunderlandSR1 3SD

T : +44 (0)191 515 2554F : +44 (0)191 515 3405

E : adrian.moore@sunderland.ac.uk