Patent Application
20190212347
The present invention relates to a specific and sensitive bio-analytical method for detection of insulin or insulin analogues in plasma, serum or any other biological fluid. It particularly relates to labelling of biological compound with stable isotopic nitrogen and bio-analytical method using solid phase extraction and liquid chromatography with tandem mass spectrometric detection.
For proteins based biological drugs, it is necessary to detect proteins such as insulin contained in the plasma, serum or any other biological fluid, and measure them quantitatively in order to elucidate the function of the protein, or for testing and screening of the protein, or to measure the systemic exposure of the protein. Therefore, if a more general method for quantifying the amount of insulin with a high sensitivity is established, it becomes possible to estimate drug kinetics during preclinical testing or in clinical trials. Thus, it would be possible to estimate drug kinetics in human at an early stage of drug development.
Typically, radioimmunoassay (RIA) or ELISA methods have been used for quantifying insulin in biological samples. Attempts to generate antibodies specific to Insulin analogue ‘IN-105’ were not successful, as the immunisation cycles yielded antibodies which showed identical binding to human insulin and IN-105, indicating that the addition of the small alkyl polyethylene glycol (PEG) does not alter the structure significantly enough to differentiate the molecule immunologically. The difference in molecular weight between the two molecules is 217 Daltons due to the alkyl PEG in IN-105. Hence, a specific ELISA based estimation assay could not be developed for IN-105.
Conventionally, a method using electrophoresis such as two-dimensional electrophoresis was conducted for quantifying biogenic proteins. With this method, detection and quantification were performed by staining the protein to be quantified, or by autoradiography, or by using an antibody specific to a particular protein (western blot). However, it was difficult to apply these methods for quantifying a high molecular protein like insulin or insulin analogue since it is not possible to electrophorese insoluble proteins or high molecular proteins.
On the other hand, there have been significant advances in the field of mass spectrometry, and the technique have been considered and used for detecting or measuring various biological materials. Typically an LC/MS/MS instrumentation involve liquid chromatography (LC) for separation of analytes, followed by ionisation chamber where the sample undergoes desolvation and ionisation and the masses are then detected by mass spectrometer. Advancements in the design of mass spectrometers supported consistent fragmentation of such a large molecule in its intact form, allowing the use of multiple reaction monitoring (MRM) mode, making the method highly specific to IN-105.
The human insulin protein is composed of 51 amino acids, and has a molecular mass of 5808 Da. It is a dimer of an A-chain and a B-chain, which are linked together by disulphide bonds. Several analogues of human insulin are available such as IN-105, Insulin Aspart, Insulin Lispro, and Insulin Glargine. These insulin analogues are closely related to the human insulin structure, and were developed for specific aspects of glycaemic control in terms of fast action (prandial insulin) and long action (basal insulin).
A specific LC/MS/MS method could be developed for measurement of intact IN-105 molecule in plasma. Sample preparations could be carried out using offline solid phase extraction of the analyte, followed by online solid phase extraction before analysis of the samples by LC/MS/MS.
The object of present invention is to provide a sensitive analytical method for the detection and quantification of insulin or insulin analogues IN-105 in human plasma at the concentration range of 0.20 ng/ml to 50.0 ng/ml.
The present invention relates to a bio-analytical method for the detection of insulin or insulin analogues such as IN-105 in human plasma.
The method comprises labelling of insulin or insulin analogue by a stable isotope of nitrogen i.e. 15N. Labelling is attained by stable isotope of nitrogen (15N) by providing labelled nitrogen in growth and fermentation medium. The cells are grown in labelled medium therefore almost all nitrogen atoms in insulin or insulin analogues are substituted with stable isotope 15N. The labelled nitrogen source used for the method is ammonium sulphate [(15NH4)2SO4].
Another aspect of the present invention relates to determination of intact molecule of the labelled insulin or insulin analogues which is further determined using solid phase extraction and liquid chromatography with mass spectrometric detection.
Present invention relates to bio-analytical method for detection of insulin or insulin analogues using liquid chromatography with tandem mass spectrometry.
In particular, the present invention relates to a highly specific method for quantification of IN-105 in biological matrix.
In one of the embodiments, biological matrix is whole blood, blood serum, blood plasma, urine, fermentation broth or buffer.
The present method allows us to determine IN-105 exposure under variety of disease conditions. Method can distinguish IN-105 from co-administered insulin/insulin analogue and endogenous insulin. Due to the use of mass spectrometry, the method can be used to determine different insulin analogues simultaneously as long as there is a difference in the molecular weight. The method can be further developed to additionally determine C-peptide levels along with insulin or its analogues. The method in general provides a useful alternative to immunological methods.
The scheme of the process is depicted in
IN-105, is a novel ‘rapid acting insulin analogue’ being developed for oral delivery as disclosed and described in U.S. Pat. No. 9,101,596. The molecule has an amino acid sequence identical to that of human insulin, except that it has been conjugated with a short chain alkyl-polyethylene glycol molecule at the ε-amino group of lysine at the 29th position of the B-chain.
Due to the high specificity of the method, IN-105 could be selectively quantified in presence of any human insulin or co-administered insulin like insulin Glargine. This makes the method very useful in determining pharmacokinetic exposure of IN-105 following oral administration as demonstrated from the pharmacokinetic profile obtained from 10 mg dosing in patients with T1D (Type 1 Diabetes).
Upstream process involves labelling of insulin or insulin analogues by stable isotopic non-metal elements such as nitrogen (15N), sulphur (34S), carbon (13C), oxygen, or hydrogen. The isotopic nitrogen (15N) source in the culture medium is at least one of ammonium sulphate, Ammonium phosphate, Ammonium hydroxide, Methylamine, Urea; the isotopic carbon (13C) source in the culture medium is at least one of Methanol, Glycerol, Glucose, Sorbitol; isotopic sulphur (34S) source in the culture medium is at least one of Calcium sulphate, Magnesium sulphate, Potassium sulphate; D20 for hydrogen and H2O18 for oxygen labelling. The scheme of the process is depicted in
Downstream process involves validation method for determining pharmacokinetic exposure in normal and disease plasma from human subjects. The validation study included linearity, accuracy and precision, selectivity and specificity, repeatability and reproducibility.
The upstream process begins with seed culture preparation, which involves inoculation of seed flasks from the culture of cell bank prepared by using a recombinant strain, Pichia pastoris. The strain carries a gene which codes for the expression of insulin precursor. The seed flasks were grown over a period to increase the cell mass which will be used as the inoculum to start the production fermenter. Fermentation is carried out in two phases viz. batch phase and fed batch phase. During batch phase, the fermentation is performed to increase the cell mass using glycerol as carbon source and during fed batch phase methanol is used to induce the secretion of insulin precursor along with 15N labelled ammonium sulphate as the only nitrogen source during process. The insulin precursor protein with labelled 15N is released into the supernatant during fermentation.
After fermentation, the downstream purification process serves to isolate the insulin precursor from the cell-free supernatant and convert it into a Methoxy-triethyleneglycol-propionyl-NεB29 recombinant insulin precursor, which through a trypsin and carboxypeptidase B catalysed reaction gets converted into insulin. Multiple HPLC steps are used to resolve close product-related impurities during this process. Multiple chromatography steps are used to resolve product related impurities. After the purification steps, the product is crystallized and lyophilized.
Estimation methods specific to an insulin analogue is useful in clinical investigations in differentiating the exogenously given insulin analogue from endogenous insulin. In the context of IN-105, a method for specifically estimating IN-105 provides unequivocal evidence for absorption of the insulin analogue upon oral delivery. The implications of such a finding are of immense importance in clinical development of oral insulin. IN-105 being a rapid acting insulin, it is expected to be co-administered with a basal insulin e.g. Glargine, under specific therapy conditions. Therefore, specificity of the LC/MS/MS method was tested in presence of basal insulin Glargine.
Upon obtaining 15N labelled product of insulin or insulin analogue, the product was used as internal standard in analytical method for determination of IN-105 in plasma. Analytical method can be used for the determination of insulin or insulin analogues in human plasma or other biological fluids containing K2EDTA or K3EDTA (either Di-potassium or Tri-potassium EDTA).
Analysis employs liquid chromatography—tandem mass spectrometry using TurbolonSpray, in positive ion, multiple reaction monitoring mode. Samples are prepared using off-line Solid-phase extraction (SPE) extraction, followed by on-line SPE in 96-well format.
The method relates to determination of insulin or insulin analogues in human plasma. Preferably, the method is employed to determine insulin or insulin analogues over the concentration range 0.200 ng/mL to 50.0 ng/mL.
Inoculum Flask: The procedure for labelling begins with seeding of cell line in inoculum flask (either directly or through seed flask) that contain medium, designed for same as shown in table 1.
When OD (Optical Density) reached to desired level (10), the inoculum from inoculum flask was transferred further for fermentation. Fermentation involved batch phase and methanol fed-batch.
Composition of fermentation medium described in table 2-4.
Biotin (0.2 g/L) was dissolved in potable water and filter sterilized through 0.2μ before use.
1. Fermentation medium was then autoclaved at 121° C. for 60 min.
2. Fermentation started with 30° C. with 350 rpm.
3. After connecting the fermenter, pH was adjusted to 4.9 with 20% w/w NaOH (sodium hydroxide) solution.
4. Sterile trace salt solution and biotin was added (4.35 ml/L of each). As soon as trace salt was added, DO was dropped.
5. 12.0 mL of trace salt solution, 12 mL of D-biotin solutions and 2.5 gm of 20% H2SO4 (w/w) are added per litre of methanol. Methanol is filter sterilized through 0.2μ before feeding into fermenter.
6. 6. Once the OD in the Inoculum flask was reached to 10-15, 250 ml of inoculum was added.
7. 7. RPM was increased from 350 to 800 in 7-8 steps.
Parameters of batch phase fermentation are
1. pH: 5.0±0.2
2. Temperature: 30±2° C.
3. Dissolved Oxygen (DO): 30%
DO was first maintained by increasing the RPM manually. When DO was reached to 40%, RPM was increased by 200 in one step up to maximum.
Once RPM was reached to maximum, DO (Dissolved Oxygen) was put in cascade of gas mix only starting with minimum of 10% and maximum of 30% and then increased as per the requirement.
When DO started to shoot up and pH started rising, sample was taken and analysis for Wet Cell Weight was done. pH was then changed to set point 6.0 whereas, temperature was adjusted to 23° C.
When batch phase was over, after one hour methanol phase was started.
Parameters of methanol fed-batch fermentation (Methanol induction phase) are
1. pH: 6.0±0.2
2. Temperature: 23±2° C.
Once the DO was raised in batch phase, methanol addition was started. (Refer table 4).
Methanol flow rate was checked periodically by using balance (0.8 density factor). Balance reading were recorded continuously.
The fermentation was checked periodically for methanol accumulation (1-5 min) during methanol induction phase as mentioned in the table 5.
The insulin or insulin analogue precursor protein with Labelled 15N is released into the supernatant during fermentation.
After fermentation, pH of final broth is adjusted to pH 2.5 with ortho-phosphoric acid followed by centrifugation to get cell free supernatant which further subjected to the downstream purification process to isolate the insulin or insulin analogue precursor from the cell-free supernatant and convert it into a Methoxy-triethyleneglycol-propionyl-N-ε-B29 recombinant insulin or insulin analogue precursor, which through a trypsin and carboxypeptidase B catalysed reaction gets converted into insulin or insulin analogue. The scheme of the process of conversion of IN-105 precursor is depicted in
Control Human Plasma (K2EDTA) from normal healthy volunteers (NHV) was obtained.
Control Human Plasma (K2EDTA or K3EDTA) from patients diagnosed with either type one (T1DM) or type two diabetes (T2DM) were also obtained. Plasma from patients with type 1 and type 2 diabetes was procured from external supplier and was available either Di-potassium EDTA (K2EDTA) or Tri-potassium EDTA (K3EDTA).
Control Human Blood (K2EDTA or K3EDTA) was obtained from volunteers for use in whole blood stability experiment and in preparation of the matrix used in the assessment of haemolysis (2% whole blood in plasma).
Lipaemic plasma was prepared by the addition of intralipid to control human plasma (K2EDTA) in a ratio 1:9, was used to assess the impact of lipaemia on the method. Haemolysed (2% whole blood in plasma) plasma was also obtained.
All the plasma obtained, either from external supplier or in-house volunteers, was stored at −20° C. prior to use and whole blood was stored at 4° C.
Two separate stock solutions were prepared by dissolving 2 mg of IN-105 and dissolving in 1 mL of 2% acetic acid containing methanol:water in the ratio of 30:70 v/v. Thus, the stock solution of 2000 μg/mL was prepared.
One was named “calibration stock” and other was named “quality control stock”. The stock solution was stored at −20° C. for 40 days.
Calibration stock solution was prepared by adding 50 μL of stock solution and diluting it in 450 μL of diluent (0.1% TFA in acetonitrile:water 50:50 v/v).
Quality control (QC) samples were prepared as per table 6 in manner having final concentration of 0.2 (LLOQ QC), 0.6 (LoQC), 20 (MeQC), 40 (HiQC) and 50 (ULOQ QC). These QC samples were stored at −20° C. and −80° C. for a period of 73 days and were used only for validation purpose.
Various standards prepared for validation study that includes analytical standard, internal standard, co-administered standard and calibration standard.
The final calibration standard concentrations were 0.2, 0.4, 1, 5, 10, 20, 45 and 50 ng/mL.
The samples obtained using labelled insulin or insulin analogue in biological matrix by off-line solid phase extraction (SPE) followed by on-line SPE in 96 well formation.
Sampling was done for sterility, wet cell weight (WCW), Titre, pH and conductivity on every day basis (every 24 hrs.).
Quality control samples prepared in quality control working solution in TFA (0.1%) in MeCN:H2O 50:50, freshly on the day of analysis.
Aliquot (300 μL each) of samples were prepared in a 2 mL 96 well plate. To these aliquots, 25 μL of internal standard (100 ng/mL) was added. None of the blanks were spiked with internal standard. The plate was vortexed for 2 minutes at 1000 rpm. To this, 50 μL of 6 M guanidine HCl was added and the plates were vortexed for 2 minutes at 1000 rpm followed by sonication for 10 minutes. To the wells, 300 μL of 10 mM tris buffer was added and plate was vortexed for 2 min at 750 rpm for mixing.
In one embodiment nitrogen isotope labelled insulin having purity 96.3% and 96.1% respectively, was used as analytical standard and internal standard for further analysis. Glargine (3.64 mg/mL) of 100% purity was used as co-administered standard in the analytical procedure. The stability studies to understand storage stability and solution stability were carried out.
In one embodiment, for stability assessment of IN-105 in whole blood, IN-105 was spiked in whole blood samples at LoQC and HiQC concentrations. The samples were sub-aliquoted and stored at room temperature and on ice. Aliquots of whole blood were taken up to 2 hrs. The samples were processed for analysis by centrifugation at 4000 rpm at 20° C. for 10 minutes. Separated plasma was harvested and stored at −80° C. until the time of analysis.
Assessment of sample stability on storage (storage stability) was carried according to table (8), Acceptance criterion was defined as a recovered concentration within ±15% of the nominal spiked concentration. All standards were used within its stability period.
System suitability was tested prior to run. The signal at the LLOQ should be at least 5 times greater than that of the noise and the chromatography and retention time should be consistent with that seen previously. Carry-over greater than 20% of the LLOQ has been observed within the validation and therefore carry-over within the system suitability samples is anticipated but should be no greater than 50% of the LLOQ when directly after the ULOQ and less than 20% of the LLOQ in the blank injection. The samples with be injected such that each type of validation sample, at each concentration level, was distributed throughout the run. Table 9 elaborates system suitability checked prior to run.
Solid phase extraction was carried out using waters Oasis Max (10 mg) SPE plate. Samples were prepared using off-line SPE extraction followed by on-line SPE. Upon obtaining labelled insulin, calibration standard and quality control standards were prepared.
Prior to each validation run, system suitability samples were analysed. These samples typically consisted of samples at the LLOQ, ULOQ and two blank matrix samples thus allowing qualitative assessment of the quality of the chromatography, sensitivity and carry over; with the exception of analytical runs 1 to 7 where only one matrix blank sample was injected after the ULOQ sample (as mentioned in table 7). Although this was a deviation from the Analytical Method, any carry-over observed was not considered to have impacted on the analytical runs.
The SPE cartridges were conditioned with methanol (300 μL) and equilibrated with 300 μL of water. Samples were loaded on to the plate, for ensuring adequate mixing they were aspirated and dispensed. Plate was washed with 500 μL 1M ammonium acetate in MeCN:water (1:10:90 v/v/v), followed by washing with 500 μL 1M ammonium acetate in MeCN:water (1:50:50 v/v/v). Packing material was dried using maximum pressure. 0.1% Triton X-100 was added to a fresh 1 mL well plate. Samples were eluated with 250 μL of 2% formic acid in MeCN:water (50:50 v/v) to the Triton X-100 containing plate. To each of these eluates 300 μL of 0.1% TFA in water was added and mixed with aspiration and dispensing. Samples were capped and taken for analysis.
Mobile phase A was TFA 0.02% in acetonitrile:water (10:90 v/v); Mobile phase B was TFA 0.02% in acetonitrile:water (90:10 v/v). Initial concentration of mobile phase A was 85% till 30 s, from 30 s to 4 min 30 s, mobile phase A changed from 85% to 60% in a linear fashion, over next 10 s mobile phase A changed to 10% and stayed at 10% till 5 min 30 s. For the equilibration, over next 10 s mobile phase A changed back to 85%. The run time was completed at 6 min 30 s. Temperature of elution was maintained at 50° C. Elution was carried out using 0.5 mL/min flow rate. Injection volume was 500 μL. Samples were maintained at 10° C. during the sequence. Wash solvent 1 was 0.01% TFA in acetonitrile:water (10:90 v/v), Wash solvent 2 was 0.01% TFA in methanol:water: Triton-X100 (70:30:0.05 v/v/v), wash solvent 3 was Ammonia (0.5% of 28%) in methanol:water (80:20 v/v).
Cartridges were conditioned using 500 μL methanol at 5 mL/min flow rate. Online cartridges were equilibrated with 500 μL of (0.1% TFA) in MeCN: water (10:90 v/v). Backflush wash was given using 1 mL of (0.1% TFA) in MeCN:water (10:90 v/v) at 2 ml/min flow rate. Following that cartridges were washed using 1 mL of (0.1% TFA) in MeCN:water (20:80 v/v) at 5 ml/min flow rate. Clamp flush 1 was carried out using 500 μL of (0.1% TFA) in MeCN:water (10:90 v/v) at 5 mL/min flow rate, clamp flush 2 was carried out using (0.1% TFA) in MeCN:water (90:10 v/v) at 5 ml/min flow rate and Clamp flush 3 was carried out using 500 μL of (0.1% TFA) in MeCN:water (10:90 v/v) at 5 ml/min flow rate.
For IN-105, 1506.9 m/z a four charge state was isolated as parent ion and its transition to 1820 m/z daughter ion was monitored. For 15N labelled IN-105, 1520 m/z was isolated and its transition to 1838.5 m/z daughter ion was monitored for quantification. The molecular weights and charge states of human insulin along with transitions monitored in the MRM mode and summarized as follows in table 10.
The validation study included linearity, accuracy and precision, selectivity and specificity, repeatability and reproducibility.
Validation consisted of intra run precision and bias, inter-run precision and bias, dilution integrity, matrix related modification of ionisation, auto-injector carry over, stability in whole blood at room temperature and on ice, freeze thaw stability of plasma over 5 cycles, 24 h room temperature stability of plasma, frozen stability in plasma at −20 and −80° C. for 73 days, recovery and extract stability at 10° C.
Additional validation runs were analysed to carry out additional experiments that would not fit within the runs used to assess precision and bias. The runs to assess precision and bias were designed to be as large as a potential study sample run (96 samples). Where additional samples were required to make a validation run as large as a study sample run, then control human plasma samples were used for this purpose.
Following table 11 elaborate parameters of validation study.
ΔInvestigated in plasma from normal healthy volunteers (NHV) & patients with T1DM or T2DM
In the first analytical run, a calibration standard which was prepared at a concentration of 40.0 ng/mL and not at 45.0 ng/mL as per the study plan. All other runs contained calibration standards at the concentrations specified within the study plan. The mean intra-run precision was found to be less than, or equal to 11.1% and the mean intra-run bias was between 0.4% and 3.0%. The inter-run precision and bias was 16.5% and 2.5% at the LLOQ QC concentration, at all other levels the inter-run precision was equal to or less than 8.9% and the inter-run bias was between 0.3% and 2.0%.
In addition to the experiments described above, selectivity of the method in plasma (K2EDTA/K3EDTA) from different sources, effect of lipaemia and haemolysis, selectivity and modification of ionisation of insulin in the presence of Glargine and stability of solutions of insulin or insulin analogues investigated.
Thorough check was performed for stability of plasma at various conditions.
For a calibration curve to be acceptable, 12 (75%) of the calibration standards had to have a back-calculated concentration of IN-105 within ±15% of the nominal value (±20% for the LLOQ). For a given calibration curve, calibration standards had to be included in the regression if the back-calculated concentration deviated from the nominal by less than, or equal to, 15% (20% at the LLOQ). A calibration standard had to be omitted from the regression if the calculated concentration of IN-105 deviated by more than 15% from the nominal (20% at the LLOQ). Up to four calibration standards (25%) could be omitted from the calibration curve, to achieve the acceptance criteria; otherwise the run was rejected. The acceptance criteria for mean intra-run and the inter-run precision was less than, or equal to, 15% at all levels, other than at the LLOQ, at which precision had to be better than, or equal to, 20%. The mean intra-run and inter-run bias had to be within ±15% of the nominal concentration at all levels, other than at the LLOQ, at which bias had to be within ±20% of the nominal concentration.
The study design for these additional validation experiments are detailed in Table 12 below:
ΔInvestigated in plasma from normal healthy volunteers (NHV)
The method was not affected by inter-individual variability, lipaemia, and haemolysis or by presence of Glargine. The observation infers that method is sufficiently accurate and precise and have sufficient selectivity and reliability that allow determination of insulin in human plasma samples over the examined range.
Insulin was found to be stable in plasma when stored at room temperature for up to 24 hours, after five freeze/thaw cycles and after 244 days storage at both −20° C. and −80° C. Also found to be stable in extracts when stored at 10° C. for approximately 85 hours.
Labelled insulin was stable in acetic acid (2%) in methanol:water (30:70 v/v) when stored at −20° C. for up to 40 days and when stored at room temperature for up to 24 hours.
Blood samples were collected in tubes containing K2-EDTA. 15N-labelled IN-105 was used as an internal reference standard (ISTD) and added to tubes after separation of serum or plasma. The ISTD-spiked samples were subjected to a mixed-mode anion exchange-reverse phase solid phase extraction process in a 96-well format. The eluate from the mixed-mode SPE are transferred to a reverse-phase (C8) SPE set up in a 96-well format. This RP SPE was setup online with an analytical reverse phase HPLC system. The eluate from the RP SPE were transferred online to the C18 analytical chromatography column and subjected to reverse phase HPLC.
The eluate from the analytical chromatography column passes into a triple quadrupole mass spectrometer, where an ESI process generates gas-phase ions from the eluate. These gas phase ions were then analysed in MRM mode as follows.
The ions pass into the first quadrupole, where ions with m/z values in a narrow range are selected. Ions within this narrow m/z range are allowed to pass through to the second quadrupole.
At the second quadrupole, intact IN-105 and ISTD molecules (and any other ions with m/z values in the same range) are subjected to a collision-induced fragmentation process in presence of an inert gas. This generates fragment ions characteristic of the parent species that were allowed to pass into the second quadrupole. These fragmented species then pass to the third quadrupole.
In the MRM mode, the third quadrupole will be set to select for ions having m/z values characteristic of fragments generated from IN-105 and ISTD. Fragments from other species that happened to have the same intact m/z as IN-105 and ISTD will be eliminated at this stage.
Only the fragments allowed through the third quadrupole are then detected as an ion current. The peak in the extracted Ion Chromatogram (XIC) for IN-105 and ISTD were both found to be integrated. The ratio of the integrated area of IN-105 is to ISTD was used as a measure of the quantity of IN-105 present in the sample.
The method was considered validated successfully since it passed all the pre-determined criteria in the validation protocol. The validated method was subsequently used for measurement of IN-105 levels in a euglycemic clamp study carried out in patients with Type 1 diabetes. The method for the determination of IN-105 in human plasma has been validated successfully over the concentration range 0.200 ng/ml to 50.0 ng/mL.
Plasma concentration data for each patient and treatment was analysed by a non-compartmental method. The area under plasma level curve for AUC0-t was calculated by the trapezoidal rule. The primary pharmacokinetic parameters (mean±SD) were Cmax, AUClast, Tmax and PD parameters were Tmin, Cmin, AUClast. Ratios and 90% CIs of geometric means were calculated for PK and PD parameters from mixed effects model with fixed effects for sequence, period and treatment, and patients within sequence as a random effect for log transformed Cmax and AUC. The maximal plasma concentration of IN-105 as well as the area under the PK curve are linearly correlated significantly (p<0.05) with the dose employed (as per table 16 below).
The average plasma concentration time profile of IN-105 obtained after administration of IN-105 tablets in volunteers is shown in
Plots of mean plasma concentration as a function of time after dosing shows the expected time profile of drug concentration in plasma. The plot for the 30 mg/kg dose is shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201641002615 | Jan 2016 | IN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2017/050303 | 1/20/2017 | WO | 00 |
