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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-10-07 to 1999-01-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
S9 homogenates of liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium, prepared from healthy male Wistar rats, were used. The rates of hydrolysis of the lactate esters by these homogenates were determined, and the enzyme kinetic parameters Km and Vmax were established, where possible.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylpropyl-(R)-2-hydroxypropanoate
EC Number:
407-770-0
EC Name:
2-methylpropyl-(R)-2-hydroxypropanoate
Cas Number:
61597-96-4
Molecular formula:
C7H14O3
IUPAC Name:
2-methylpropyl-(R)-2-hydroxypropanoate
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Isobutyl-R-lactate
- Analytical purity: 99 %
- Lot/batch No.: R(+)IBL-2
- Storage condition of test material: Ambient temperature
Radiolabelling:
no

Test animals

Species:
other: Homogenates of rat liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium.
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
S9 homogenates of liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium, prepared from healthy male Wistar rats were used. The esterase activities of the various homogenates towards the model substrate p-nitrophenyl-butyrate were determined according to the method described by Bogdanffy et al. (1987):
100 pM p-nitrophenylbutyrate and a suitable amount of homogenate were incubated in 0.1 M phosphate buffer pH 7.8 at 25 °C in a total volume of l mL. The rate of hydrolysis was measured spectrophotometrically at 400 nm.

Administration / exposure

Route of administration:
other: Incubation with homogenates of rat liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium.
Vehicle:
other: 0.10 M potassium phosphate buffer pH 7.4
Details on exposure:
General incubation conditions:
The lactate esters were incubated at 37 °C in 1 mL incubation mixtures containing 0.10 M potassium phosphate buffer pH 7.4. The chemical hydrolysis was determined in incubation experiments without homogenates. Blanks were homogenates without test substances.
Incubations were performed in open tubes. The reaction was terminated by addition of 3 mL of ice-cold ethanol. After placing the samples in the
freezer for at least 20 minutes, the tubes were centrifuged for 10 min. at 4300 × g (room temperature) and decanted into new tubes. The samples
were evaporated to dryness with nitrogen and stored at < -15 °C until analysis.

Analysis:
The liberated amount of L-lactic acid was determined using the Boehringer test for the determination of L-lactic acid in foodstuffs and
other materials. The liberated amount of D-lactic acid (syn. R-lactic acid) was determined by using the Boehringer test for the determination of D- and
L-lactic acid in foodstuffs and other materials. Instead of the glycylglycine buffer (pH 10) included in the kit, a 0.1 M potassium phosphate buffer pH 7.4 (assay buffer) was used, in order to minimize hydrolysis of the lactate esters during the measurement of the liberated amounts of L- and D-lactic acid.
The incubation scheme was as follows:
Samples: 1 mL assay buffer, 1 mL demineralised water, 200 µL nicotinamide-adenine dinucleotide (NAD), 20 µL glutamate-pyruvate transaminase (GPT)
Standards: 1 mL assay buffer, 1 mL standards (lactid acid standard solution in the range of 0-450.7 nmol L-lactic or D-lactic acid), 200 µL nicotinamide-adenine dinucleotide (NAD), 20 µL glutamate-pyruvate transaminase (GPT)
The absorbance of the samples and standards were measured at 340 nm (A1). Afterwards, 20 µL L-LDH/D-LDH (lactate dehydrogenase) was added to all samples. 1 hour later, the absorbance was measured at 340 nm (A2). The detection limit of the method was arbitrarily fixed at 10 nmol (adsorption ca. 0.02). By calculating A2-A1 the increase in absorbance due to the formation of L-lactic/D-lactic acid was determined.
Duration and frequency of treatment / exposure:
The test item was incubated with the various homogenates for 5, 10, 20, 40 and 120 min. Chemical hydrolysis was measured by incubating the substrates without homogenates during 120 min.
Doses / concentrations
Remarks:
Doses / Concentrations:
The test item concentrations used were 50, 100, 250, 500 and 1250 µM.
Control animals:
other: Chemical hydrolysis was measured by incubating the substrates without homogenates during 120 min.
Positive control reference chemical:
The esterase acitivities of the various homogenates towards the model substrate p-nitrophenylbutyrate were determined.
Statistics:
The amounts of L-lactic acid or D-lactic acid (depending on the ester) formed during the incubations were calculated from the respective standard curves. The rates of hydrolysis were corrected for the chemical hydrolysis, which was assumed to be a linear chemical process. The initial rates of hydrolysis were calculated from the amounts of
liberated L-lactic acid/D-lactic acid (corrected for chemical hydrolysis) with the regression model:
liberated lactic acid (nmol) = a.(time) + b.(time)²
or with the regression model
Liberated lactic acid (nmol) = a.(time),
with: a = regression coefficient of the linear component
b = regression coefficient of the quadratic component
The regression coefficient a represents the initial rate of hydrolysis expressed as nmol/min. After calculating the initial rates of hydrolysis expressed as nmol/min/mg S9 protein, the enzyme kinetic parameters Km and Vmax were determined by the curve-fitting program EZ-FIT™ (version 4.13 for MS Windows).

Results and discussion

Preliminary studies:
The protein concentrations, and the esterase activities towards the model substrate p-nitrophenylbutyrate of the various tissue homogenates is presented in Table 1.
Main ADME results
Type:
metabolism

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Chemical hydrolysis of isobutyl-R-lactate:
No detectable amount of lactate formed by chemical hydrolysis was measured after 2 hour incubation.

Initial rates of hydrolysis of isobutyl-R-lactate:
The results are presented in Table 2.

Enzyme kinetic parameters for the hydrolysis of isobutyl-R-lactate:
Enzyme kinetic parameters Km and Vmax were calculated from the initial rates of hydrolysis (table 2). The results are presented in Table 3.

Calculated times in which at 99 % of isobutyl-R-lactate would be hydrolysed:
In order to extrapolate the obtained kinetic parameters in terms of disappearance rates of lactate esters in the organs/tissues, the obtained kinetic parameters were scaled up to hydrolysis rates expressed per weight of tissue, by using the total amount of protein per gram of tissue. Subsequently, the disappearance in time of the ester compounds in the organs/tissues was calculated by the Michaelis-Menten or first order equation. A starting concentration of 500 µM was used. However, it had to be assumed that the equilibrium of the reactions are completely on the side of the hydrolysed compounds. The calculated disappearance rates would be higher in vivo. The times were calculated in which at least 99 % of the ester would be hydrolysed. Results are presented in Table 4. Based on the results it can be stated, that isobutyl-R-lactate is hydrolysed very rapidly (from 3.5 to 255 seconds, depending on the tissue).

Any other information on results incl. tables

Table 1: Mean protein concentrations and esterase activities towards p-nitrophenylbutyrate of the various tissue homogenates
Homogenate Protein concentration (mg/mL) Esterase activity towards p-nitrophenylbutyrate
Amount of protein used in assay (µg) Activity
(µmol/min/mg protein)
 
Nasal olfactory 7.14 11.9 1.013 ± 0.028
Small intestinal mucosa 9.62 2.41 7.836 ± 0.064
Liver 22.3 11.2 0.852 ± 0.010
Skin 2.52 63.0 0.113 ± 0.003
Blood 41.6 104 0.0134 ± 0.0001

Table 2: Initial rates of hydrolysis of isobutyl-R-lactate. Enzymatic activities are expressed as nmol/min/mg protein
Lactate ester Concentration
(µM)
Initial rates of hydrolysis
Nasal olfac. epithelium Small intest. Mucosa Liver Skin Blood
 
isobutyl-R-lactate 50 47 5.1 56 4.8 -
100 85 9.4 89 8.9 0.3
250 177 15.1 183 19.3 1.6
500 262 24.7 274 27.7 3.5
1250 308 42.8 409 41.9 7.7

- = no detectable rate of hydrolysis

Table 3: Enzyme kinetic parameters for the hydrolysis of isobutyl-R-lactate. Vmax is expressed as nmol/min/mg protein

Homogenate Kinetic parameter Isobutyl-R-lactate
 
Nasal olfactory Km (µM) 313 ± 57
Vmax 396 ± 28
Small intestinal mucosa Km (µM) 923 ± 210
Vmax 73.6 ± 9.0
Liver Km (µM) 557 ± 32
Vmax 589 ± 16
Skin Km (µM) 569 ± 40
Vmax 60.7 ± 2.0
Blood Km (µM) first order
v = 0.00626 × S

Vmax

v = rate expressed as nmol/min/mg protein; S = ester concentration

Table 4: Calculated times (seconds) in which at least 99 % of the test item would be hydrolysed
Homogenate Isobutyl-R-lactate
 
Nasal olfactory epithelium 5.0 s
Small intestinal mucosa 87 s
Liver 3.5 s
Skin 140 s
Blood 255 s

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: Isobutyl-R-lactate is hydrolysed effectively in rat liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium.
Isobutyl-R-lactate is hydrolysed effectively before, during or immediately after absorption, even at high concentrations, in rat liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium under the formation of D-lactic acid.
Executive summary:

The rate of hydrolysis (both abiotic and enzymatically catalysed) of isobutyl-R-lactate (99 % purity) by homogenates of rat liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium was investigated in vitro. The test item was incubated with the various homogenates for 5 ,10, 20, 40 and 120 min at concentrations of 50, 100, 250, 500 and 1250 µM.The enzyme kinetic parameters Km and Vmax were calculated from the initial rates of hydrolysis.

Taking into consideration the enzymatic parameters of the various homogenates, it can be concluded that isobutyl-R-lactate will be effectively hydrolysed before, during or immediately after absorption, even at high concentrations. The 99 % degradation times vary between 5.0 and 255 seconds, depending on the type of tissue.