Chemometrics Coursework,

February 2010
A summary review of chemometric publication
1. You are provided with two Chemometric publications. Choose one, read and answer the
following:
a. Write about the background to the publication (one paragraph)
b. Write about the basic theory of the instrument used to collect the data (one
paragraph include diagram if necessary)
c. Identify the type of data generated (Include sketches, diagrams and tables where
necessary)
d. Comment on the main chemometric methods or tools used to analyse the data
(Include equations, diagrams and tables where necessary. Not more than one or two
paragraphs)
e. Identify two main references in the publication considered to be important for the
chemometric data analysis methods used
f. What was the main conclusion or findings
g. List two possible Advantages and Disadvantages of the approach used in this
publication
Note:
i. Coursework should be no more than 7 pages including figures and tables
ii. Report layout, must be in the format should have sub headings a to g above. Include your
Name and Student ID number
A summary review of chemometric publication
Uchenna Onyugwu.
000397559.

A:
Counterfeits make up to more than 10% of the global medicines market according to
the United States Food and Drug Administration (FDA) and are a continuing problem
for the developed as well as underdeveloped countries. Evident to this is the growth
of counterfeit drug cases (58 in 2004 and 9 in 1997) according to the FDA’s Office of
criminal investigations. Examples of drugs counterfeited include hormones and
cancer drugs. Viagra and Cialis are also counterfeited widely and in developing
countries malaria and tuberculosis drugs. Such drugs contain insufficient/or no
active ingredient (API) and/or false packaging. As a result analytical control is now of
equal importance to quality control in the production department. Chemical analysis,
after visual check, is via High performance Liquid Chromatography (HPLC) as a
quantitative test. UV spectroscopy, near- infrared and Thin layer chromatography
have been used in recent years for quality control.

B:
Fast analysis of fake and original artesunate tablets by Raman spectroscopy. Non-
invasive, fast analysis and no sample preparation makes Raman analysis viable in
counterfeit drug analysis. By using a combination of chemometric algorithms
(compare recorded spectra with library database) and automated instrument
operation with spectral analysis Raman can be used in detecting counterfeits. A
spectrum of pure artesunate was recorded as were suspect artesunate. All 3
(B,C,and D) were compared to A with presence or absence of raman bands
(functional groups) used to detect fakes (see fig.2). A combination of colorimetry and
LC-MS were used to confirm the results of raman analysis. LC-MS was used as a
reference point for raman data retrieved.

C:
The type of data retrieved by raman analysis were simple vectors as shown in table
one. Chemometric classification produced subsequent data in Fig 3-5. Figure 3 data
was retrieved via data matrix, mean, zero mean and unit variance (SNV). Giving
scree, loading and score plots. Figure 4 is a Dendrogram obtained via specral
means/averages. Figure 5 is a raman spectrum with simple vectors.
D:
Principal component analysis (PCA) with subsequent HCA ( hierarchical cluster
analysis) was performed to give an automated classification algorithm. A data matrix
was built prior to analysis (50 rows and 1467 columns) by first acknowledging
inhomogeneity of sample by taking the mean of triplicate spectra in triplicate for each
sample. Savitsky-Golay algorithm, 13 data point window and second degree
polynomial was used to correct nonspecific fluorescence then each first derivative
spectrum was scaled to unit variance and zero mean, then PCA algorithm was run
on the data matrix made (see fig.3 for results). Compartmentalisation in group C is
accounted for by PC 4 scores, PC 2 and 3 are discriminate of group D spectra. HCA
was done by using scores for the first 4 principal components to input K-means
clustering algorithms based on the Euclidean distances as shown in fig.4. Only 4
principal components were used to prevent over-fitting.

According to fig 4 in the original artesunate group, a spectrum is very different to the
others as shown in (*) as it had low wavenumber raman bands (see fig 5) caused by
rutile, a polymorph of TiO 2 and is calssified into the correct group by the algorithm.
A low degree of crystalinity is shown by broader Raman bands exhibited by 4
artesunate samples in the A- branch of the dendrogram. For more complex database
reference more advanced techniques like ANNs (artificial neural networks) can be
used. Qualitative composition was of primary focus rather than quantitative in times
past but this technique does both.

E:
 Savitzky A, Golay MJE. Anal. Chem. 1964; 36(8): 1627.

 Edwards HGM, Hassan NFN, Middleton PS. Anal. Bioanal.Chem. 2006;

384(6): 1356.

F:
The combination of multivariate clustering and Raman spectroscopy differentiates
vividly original from fake tablets and also produce a ‘fingerprint’ (chemical) of
different types of counterfeits in order to determine relationship between different
tablets. The combination above can help in forensic field in finding the trade routes
and sources of counterfeit drugs.
G:
ADVANTAGES:

1. Fast (lack of sample preparation) and reliable method for characterisation of

genuine and counterfeit antimalarial tablets.
2. Non destructive property makes it suitable for tablets. No loss of product
means more money (cost effective).

DISADVANTAGES:

1. Fluorescence interference.
2. Analytical sensitivity in quantitative analyses is not good enough.
CLINICAL USE AND EFFICACY OF ACETYLCHOLINE-ESTERASE
INHIBITORS (AChEI) IN THE TREATMENT OF ALZHEIMER’S
DISEASE(AD).
Understanding the pathology of AD is a key step in the race to discover therapeutic
macromolecules against AD. Excessive amounts of amyloid plaque and
neurofibrillary tangles in the brain, including toxic amyloid beta (Aβ) protein
aggregation is said to be a primary cause of AD. Therapeutic agents such as
donepezil, galantamine and rivastigamine are AChEIs used in treating AD because
of rising pharmacological and neuro-chemical evidence that suggest cognitive fall in
AD is linked to cholinergic deficiency. This essay review discusses all 3 drugs with
reference to their mechanism of action, clinical trial data, clinical efficacy, cost and
finally government recommendations and reasons behind them. Evidence of
neuroprotection is also looked at.

SCIENCE BEHIND NEUROPROTECTIVE ACTION.

Studies of donepezil in O2 –glucose deprivation model of ischemia using rat PC12

cells and rat primary neuronal culture of the cerebral cortex lead to evidence of
neuroprotection by AChEIs as well as neuroprotection against glutamate
neurotoxicity in a concentration dependent manner from 0.1-10μM. Pretreatment
with donepezil for 24hrs tremendously inhibited glutamate-induced loss of viability
and all 3 were also observed to protect against apoptosis. The above concentrations
were higher than their half-maximal inhibitory concentrations, meaning
neuroprotection was not due to AChEIs only. nAChR antagonists inhibit glutamate
neurotoxicity protection. Antagonistic behaviour of dihydro-β-eythroidine and
methyleaconitine (inhibit α4 and α7 nAChRs respectively) means they (receptors)
participate in donepezil and galantamine induced neuroprotection. Mecamylamine
inhibits rivastigmine instead of the subtype antagonist meaning some unseen nAChR
subtypes are participants in neuroprotection. P13K-Akt-Bcl-2 pathway involves
AG490, LY294002,PP2 and wortmannin inhibitors of Fyn, JAK2 and P13K
respectively greatly inhibited donepezil /galantamine neuroprotection but not
rivastigmine. Akt phosphorylation level was obsereved to rise after treatment with
donepezil and galantamine but not rivastigmine. Bcl-2 trancsript expression also
increased.

To sum up nAChRs is facilitated galantamine and donepezil especially α7 nAChR

directly or indirectly and activates P13K-Akt-Bcl-2 pathway to increase neuronal
survival whilst rivastigmine nAChR subtypes action are unclear.
Mechanism of Upregulation of nAChRs by AChEIs

Chronic treatment over 4 days on primary cultures of rat cortical neurons with
donepezil is independent of mRNA levels of α4 and α7 subunits seen via reverse
transcription polymerase chain reaction. Meaning nAChR is ypregulated via post-
translational mechanisms by donepezil treatment. α7 nAChR up-regulation is
inhibited after chronic donepezil treatment as well as AG 490, PP2 and LY294002
meaning α7 nAChR-P13K pathway participation. Hence up-regulation of nAChR
may be because of increase in nAChRs at the cell surface. Allosteric modulation of
nAChR function is suggested to cause up-regulation via galantamine action.

DONEPEZIL.

Clinical trial data and Efficacy.

Qualified patients were 55yrs and over, local and satisfied the following inclusion
criteria:

1. Never been on AChEI treatment prior to trial.

2. Recently diagnosed with probable AD according to DSM-IV and NINCDS-
ADRDA criteria.
3. Written informed consent by their lawyer or themselves.
4. Lack of severe disease in the last 6 months and neurological disease,
delirium, substance abuse and psychiatric disorders.

The clinical effectiveness of donepezil is such that monotherapy of the drug in

patients causes cognition measured via M@T and MMSE remains stable at 6
months, while functionality (standard of living) greatly diminishes according to
measurements via ADFACS. Baseline of MMSE ≥21 suggests that the merits of
donepezil treatment benefits the mild AD group more than the moderate AD group
(MMSE below 21). There is a slower decline (IADL), MMSE orientation improves as
do both language and memory domains as well as semantic memory domains and
M@T temporal orientation. These data therefore propose that M@T is useful in
assessing progression in early AD and that donepezil provides greater benefits in
mild compared to moderate AD.

In mild to moderate AD very few patients treated with donepezil showed

symptomatic worsening compared with patients on placebo. However unresponsive
patients showed less cognitive decline than placebo meaning more benefits to
treated patients. However according to The NICE Committee after about 4200
randomised controlled trials, the evidence from global and cognitive outcome
measurement scale studies suggest that though beneficial (donepezil) in treating AD
the standard of living and behavioural symptoms were unsatisfactory as were short
term benefits. Retrospective responder analyses models and subgroup analyses
based on severity of cognitive impairment carried out by manufacturers show more
advantages for more severe AD patients.
 Evidence of Neuroprotection.

Evidence of neuroprotection against neuronal cell apoptosis means donepezil

ameliorates progressive mental decline. Neuroprtective effect against exposed to
O2-glucose deprivation (A), on Aβ toxicity(B) and NMDA excitotoxicity(C).

In (A) donepezil exhibited a concentration dependent effect and greatly reduced LDH
release even in very small amounts (0.1μM) (Table1) and vice-versa for
galantamine. IC50 values of 0.7±0.35nM for dopenezil and 1200±33nM . This means
greater inhibitory activity on rat brain AChE by donepezil over galantamine.
Rivastigmine (0.1-10μM) did not greatly reduce LDH release but boasts good AChEI
activity. Pre-treatment with donepezil greatly reduced the amount of LDh released
against Aβ-induced neurotoxicity in a concentration dependent manner (Table2)
meaning donepezil neuroprotection is not down to AChEI activity.

In (B) neuroprotection via antagonistic effects on NMDA receptors explains

donepezil protection on rat cortical neuron cultures from NMDA induced cytotoxicity
in a concentration dependent way at 0.1μM and over.(Table1). Binding of ligands to
polyamine and ligand sites on NMDA receptor is inhibited by donepezil but
neuroprotection is solitary to direct binding to glutamate receptors.

From (B) donepezil inhibits Aβ1-40 accumulation caused by human recombinant

AChE, since it interacts with PAS (peripheral anionic site) and blocks aggresgation
by 22%. All three findings show that donepezil possesses neuroprotective effects
and improves cholinergic grain function whilst decreasing progressive
neurodegeneration in AD.

Cost Effectiveness: though manufacturers an incremental cost effective saving of

£14,000 per yr outside of the severe state, the assessment group added mortality
risk as well as other costs. Ultimately the incremental cost effectiveness rose to CQG
(quality-adjusted life year) £45,000.

GALANTAMINE

A plant alkaloid used in treating AD. Using primary rat cultured cortical neurons, the
effects of galantamine on Aβ-enhanced glutamate toxicity was studied as were
allosteric potentiating ligands for nAChRs.
Mechanism of action.

Aβ-enhanced glutamate toxicity against neurons was blocked by nicotine and/or

galantamine though inhibition was blocked by α7 nAChR antagonists. Even though
Akt phosphorilation is induced by galantamine P13K inhibitors stopped galantamine
protection and Akt phosphorilation. FKL antibody decreased galantamine’s protective
effects and Akt phosphorilation and α7 nAChR inhibition via RNA interference
reducing Akt phosphorilation. Therefore galantamine neuroprotection is partly due to
α7 nAChR-P13K pathway. In rat 6-hydroxydopamine (6-OHDA) induced
hermiparkinsonian model 6-OHDA and/or galantamine and/or nicotine administered
into the unilateral substantia nigra showed that nicotine and galantamine together
inhibited synergistically 6-OHDA induced neuronal loss and rational behaviour.
TH-immuno± neurons in the SNpc contain α7 nAChR and reduction of these neurons
was reduced by a combination of galantamine and nicotine. Hence through allosteric
modulation galantamine plus nicotine synergistically increases neuroprotection.

Evidence of Neuroprotection and Science Behind Action.

Oxidation of lipids, sugars, proteins and nucleic acid is suggested to cause neuronal
dysfunction and occurs early in AD (ROS). 2.5μM of galantamine (as confirmed in
other invivo and invitro studies) avoided AChR activity augmentation caused by Aβ.
2.5μM of galantamine inhibited both young and aged Aβ 1-40 induced
neurodegeneration. The 2.5μM of galantamine has clinical relevance and in low
concentrations of 1μM agonistically induced nAChR activity rises and decreases in
high concentrations. Therefore neuroprotection could be carried out by other non-
direct nAChRs mechanisms that are Aβ influenced. In this in-vitro study old and
young Aβ1-40 solutions used (due to suggestions that different aggregates of Aβ
cause different effects on neurodegeneration, oxidative stress, calcium influx)
caused similar degrees of neurodegeneration. Hence incubation of neuronal cells
with such solutions causes apoptosis. (Fig2).

After a 5 day incubation period Aβ aggregate formation was greatly reduced in

cultured neuron samples containing galantamine (Fig3). Compounds like
galantamine that inhibit Aβ oligomerisation have neuroprotective effects as do
various antioxidants (inhibit Aβ fibrilisation). Besides neuroprotection galantamine
inhibits lipoperoxidation (Aβ1-40 induced) and ROS formation, hence galantamine is
an antioxidant too. It decreases calcium influx caused by nAChR action. Nicotinic
receptor activity controls Calcium influx. In fig 6 we can see the galantamine
inhibition of decrease in GSH levels (caused by Aβ 1-40) in cortical neurons. This fall is
probably due to GRed and GPx function reduces in Aβ treated neurons and was
greater with new Aβ than old because of GRed inhibition. The more severe the AD
the greater the levels of GPx and GRed mRNA in the brain.
Clinical Trial Data and Clinical Efficacy.

After 6 months of galantamine therapy 81% of a population of older patients (mean

age 83.6yrs) in SERAD study similar to placebo group (completed trail) and 87% of
which tolerated highest doses of 24mg/day (Serad patients) showed less deaths and
adverse effects. Safety and tolerability were equal or above reported trails of drug in
mild-moderate AD and trials of other ChEIs with patients in severe AD. The severe
impairment battery (SIB), showed a statistically significant merit of galantamine
compared to placebob and the effects were as great as those in donepezil and/or
memantine trials with the same population.

Minimum data set activities of daily living (MFS-ADL) score measures patient’s state
with respect to standard of living and was worsened by 1-3 points from baseline
score (13-1[SD 7.7] points) whilst placebo group worsened by 1-7 points, baseline
(14-0[8.1] points). Over 6 months the change was very little and variables were
overboard to detect good beneficial effects as in Alzheimer’s Disease Cooperatrive
Study-ADL inventory (ADCS-ADL) in 2 memantine trials and with DAD scale in the
moderate-severe AD donepezil study. Placebo group had less psychiatric
cormobidities than placebo groups at baseline. Also galantamine may not improve
activity in ADL in this population hence this trial failed. Evidence from this trial
suggests that galantamine is tolerable and safe in this SERAD population and
possesses some beneficial effects although clinical efficacy is not clear due to
negative results in living standard as measured via ADL.