lipase plays a key role in dietary fat absorption by hydrolyzing triglycerides to monoglycerides and free fatty acids . as such
orlistat is a potent lipase inhibitor that is sold as a weight loss aid in prescription and over - the - counter formulations .
it has been demonstrated that orlistat is an irreversible inhibitor that covalently binds to serine 152 residue of lipase , which causes a pronounced in vitro as well as in vivo inhibition of gastric and pancreatic lipases .
previous studies on this time dependent inhibition have used two - phase emulsion monitoring activity with a ph stat , which limits sample throughput .
several studies have also revealed the significant effect of modifying the formulation of the orlistat on lipase inhibitory activity .
it was found in one study that nano - sized particles of orlistat can be used for enhanced in vitro dissolution rate and lipase inhibition .
however , to optimize the formulation for pharmacodynamic effect would require different time - consuming and costly clinical studies .
hence , there is a need for a simplified in vitro method to guide the selection of a suitable composition of formulation to optimize the release profile for obtaining better pharmacodynamic effect of orlistat products . to address this need , a dynamic in vitro single - phase system
this system was able to provide a simulation of the in vivo lipase activity and is capable of directly testing dissolution or gastrointestinal ( gi ) fluid samples to assess lipase inhibition by orlistat . to validate the developed assay , two different formulations of orlistat
porcine pancreatic lipase ( type ii ) ( ec 3.1.1.3 ) , sodium deoxycholate , sodium phosphate monobasic , isopropanol and p - nitrophenyl palmitate ( pnpp ) were purchased from sigma aldrich ( usa ) .
uv - transparent 96-well plates were used for colorimetric quantification on a tecan infinite m200 spectrophotometer .
since the majority of triglyceride hydrolysis is done by pancreatic lipase , porcine pancreatic lipase was used as a model enzyme .
crude lipase was dissolved in reaction buffer ( 10 mg / ml ) and centrifuged at 7000 g for 10 minutes to remove insolubles .
lipase assays were performed in a 96-well , clear , flat bottomed plate with 200 l reaction volume .
pnpp was used as a substrate with a reaction buffer of 50 mm sodium phosphate , 5 mm sodium deoxycholate , and 10% isopropanol at ph 8.0 [ 9 - 11 ] .
lipase assays used a 200 l reaction volume and substrate conversion was monitored with a tecan infinite m200 spectrophotometer at 410 nm .
all assays were run at 37c and reported results are the average of six replicates that were blank subtracted . to characterize the system ,
enzyme linearity of was shown by finding the initial rate for increasing enzyme concentrations . for determination of the kinetic constants ,
enzyme concentration was fixed at 0.01 mg / ml ( the lipase concentration was determined per an established calibration curve , data not shown ) and the substrate concentration was varied .
minimization of the sum of squares gave a fit to the michalis - menten equation .
pre - incubation and co - treatment studies were performed to verify single phase irreversible binding . for pre - incubation
, the lipase and orlistat were added together and incubated at 37c for varying times before substrate was added and the initial rate ( < 2 minutes ) was recorded . for co -
treatment , the substrate and orlistat were added together and the rate was monitored continuously . to find the activity as a function of time , the derivative of a 2 order polynomial fit was taken to find the instantaneous slope , which was normalized to the positive control .
two solid dosage forms of orlistat were compared using this pnpp assay , formulation 1 and formulation 2 .
a hanson usp ii dissolution apparatus was used to determine release profiles of the formulations .
the dissolution media used was 3% sodium lauryl sulphate ( sls ) , ph 6.0 at 37c .
2ml samples were manually withdrawn and filtered with a 0.45 m poly tetra fluoro ethylene ( ptfe ) filter , discarding the first ml of filtrate .
samples were analyzed on an agilent 1100 hplc with a waters nova - pak c18 3.9150 mm , 4  particle size hplc column to determine the active concentration .
porcine pancreatic lipase ( type ii ) ( ec 3.1.1.3 ) , sodium deoxycholate , sodium phosphate monobasic , isopropanol and p - nitrophenyl palmitate ( pnpp ) were purchased from sigma aldrich ( usa ) .
uv - transparent 96-well plates were used for colorimetric quantification on a tecan infinite m200 spectrophotometer .
since the majority of triglyceride hydrolysis is done by pancreatic lipase , porcine pancreatic lipase was used as a model enzyme .
crude lipase was dissolved in reaction buffer ( 10 mg / ml ) and centrifuged at 7000 g for 10 minutes to remove insolubles .
lipase assays were performed in a 96-well , clear , flat bottomed plate with 200 l reaction volume .
pnpp was used as a substrate with a reaction buffer of 50 mm sodium phosphate , 5 mm sodium deoxycholate , and 10% isopropanol at ph 8.0 [ 9 - 11 ] .
lipase assays used a 200 l reaction volume and substrate conversion was monitored with a tecan infinite m200 spectrophotometer at 410 nm .
all assays were run at 37c and reported results are the average of six replicates that were blank subtracted .
for determination of the kinetic constants , enzyme concentration was fixed at 0.01 mg / ml ( the lipase concentration was determined per an established calibration curve , data not shown ) and the substrate concentration was varied .
minimization of the sum of squares gave a fit to the michalis - menten equation .
pre - incubation and co - treatment studies were performed to verify single phase irreversible binding . for pre - incubation , the lipase and orlistat were added together and incubated at 37c for varying times before substrate was added and the initial rate ( < 2 minutes ) was recorded . for co - treatment , the substrate and orlistat were added together and the rate was monitored continuously . to find the activity as a function of time , the derivative of a 2 order polynomial fit was taken to find the instantaneous slope , which was normalized to the positive control .
two solid dosage forms of orlistat were compared using this pnpp assay , formulation 1 and formulation 2 .
a hanson usp ii dissolution apparatus was used to determine release profiles of the formulations .
the dissolution media used was 3% sodium lauryl sulphate ( sls ) , ph 6.0 at 37c .
2ml samples were manually withdrawn and filtered with a 0.45 m poly tetra fluoro ethylene ( ptfe ) filter , discarding the first ml of filtrate .
samples were analyzed on an agilent 1100 hplc with a waters nova - pak c18 3.9150 mm , 4  particle size hplc column to determine the active concentration .
the enzyme demonstrated a high degree of linearity for the range of concentrations considered ( 0 - 2.5 mg / ml ) as shown in figure 1 .
lipase - pnpp single phase catalysis exhibits michalis - menten kinetics with a km=2.7  0.2 m and kcat=0.019 s as shown in figure 2 .
lipase inhibition assays showed that orlistat is a potent , time dependent inhibitor in single phase solutions . in the pre - incubation study , an exponential decrease in the observed initial rate of the enzyme
co - treatment inhibition kinetics showed a linear decrease in the enzyme activity with time ( figure 4 ) . for the incubation results ,
all 2 order fits had a very good fit ( r>0.99 ) , which provided linear decreases in the activity with time .
lipase activity was completely negated with 20 ng / ml orlistat in a sample with 0.1 mg / ml lipase .
two different formulations were able to be distinguished in the dissolution test under identical conditions , with formulation 1 showing a faster initial release rate .
the effects were most notable with the formulation 2 , where small differences in the initial rate of release correspond to significantly higher inhibition ( figure 5 ) .
the enzyme demonstrated a high degree of linearity for the range of concentrations considered ( 0 - 2.5 mg / ml ) as shown in figure 1 .
lipase - pnpp single phase catalysis exhibits michalis - menten kinetics with a km=2.7  0.2 m and kcat=0.019 s as shown in figure 2 .
lipase inhibition assays showed that orlistat is a potent , time dependent inhibitor in single phase solutions . in the pre - incubation study , an exponential decrease in the observed initial rate of the enzyme
co - treatment inhibition kinetics showed a linear decrease in the enzyme activity with time ( figure 4 ) . for the incubation results ,
all 2 order fits had a very good fit ( r>0.99 ) , which provided linear decreases in the activity with time .
lipase activity was completely negated with 20 ng / ml orlistat in a sample with 0.1 mg / ml lipase .
two different formulations were able to be distinguished in the dissolution test under identical conditions , with formulation 1 showing a faster initial release rate .
the effects were most notable with the formulation 2 , where small differences in the initial rate of release correspond to significantly higher inhibition ( figure 5 ) .
the study demonstrated the utility of an in vitro single phase assay that would be able to directly test gi and/or dissolution samples to determine lipase activity and inhibition by orlistat .
the results confirmed and expanded the findings of previous two - phase studies that orlistat is an irreversible , time - dependent inhibitor of lipase [ 1,3,5,12 - 14 ] , despite using a single - phase reaction . in two phase emulsion studies , coalescence of emulsion particles may gave rise to reduced surface area to the point where reaction rates may become limited in part by substrate .
in addition , the resulting heterogeneity caused by  creaming  of the emulsion may compromise the ability to procure representative subsamples for conducting the lipase assay .
therefore , the developed single phase assay is arguably better method to evaluate lipase activity .
enzyme linearity over a range of physiologically relevant concentrations and adherence to michalis - menten kinetics provide a simple and robust system for testing dissolution or gi samples .
additionally , the resolution that this assay provides can distinguish between minor differences in enzyme inhibition . the ability to directly evaluate dissolution samples
is validated by the correlation with the hplc release profile . in combination with bio - dissolution system
, the method could be developed as a bio - method to determine the suitability of formulation ( such ir or sr ) for orlistat - type drugs .
a potential example of this is sucrose ester , which alters the surface tension of micelles in an emulsion . as such
, its effects would not be able to be seen in a single phase assay .
optimizing excipient formulations may allow for lower dosages of the active , but will require improved in vitro - in vivo correlations ( ivivc ) to predict pharmacodynamic behavior before human trials .
future studies using this assay with a biorelevant dissolution method and human lipase or gi fluid will verify the sensitivity of the assay in addition to providing a more accurate ivivc .
we have developed a very sensitive , single - phase assay for orlistat that mimics the time - dependent inhibition seen in emulsions . additionally , we have shown a successful proof of concept for using dissolution samples in an in vitro assay as a method of estimating in vivo efficacy .
 purposeto develop a bio - assay that would be able to directly test gastrointestinal and/or dissolution samples to determine lipase activity and inhibition by orlistat.methodsenzyme assays were performed with porcine pancreatic lipase and para - nitrophenyl palmitate ( pnpp ) in ph 8.0 reaction buffer at 37c . 
 substrate hydrolysis was monitored by absorbance changes at 410 nm . 
 the dissolution of two orlistat formulations was tested with a usp ii apparatus . 
 samples were hplc analyzed to determine release profile in addition to being diluted and directly assayed for inhibitory effect.resultsthe lipase - pnpp system demonstrates linearity and michalis - menten kinetics with a km=2.7  0.2 m and kcat = 0.019 s1 . 
 orlistat showed highly potent and time dependent inhibition with 5 ng / ml effecting 50% activity after 5 minutes in the lipase - pnpp system . 
 dissolution studies showed a correlation of the drug release profile to the inhibitory effect of dissolution samples in the assay.conclusionsthe lipase - pnpp method can be used as an in vitro assay to monitor orlistat inhibition from drug release or dissolution samples . 
Introduction
Materials and Methods
Materials
Lipase preparation
None
Estimation of enzyme kinetics
Estimation of time dependent inhibition
Dissolution studies
Results
Enzyme kinetics
Lipase inhibition
Dissolution studies
Discussion
Conclusions