FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
METHOD OF DETERMINING BIOEQUIVALENCE OF A GENERIC LINACLOTIDE PRODUCT TO THE ORIGINAL LINACLOTIDE PRODUCT
COMPLETE SPECIFICATION
(See section 10; rule 13)
SUN PHARMACEUTICAL INDUSTRIES LTD.
A company incorporated under the laws of India having their office at SUN HOUSE, 201B/1, WESTERN EXPRESS HIGHWAY, GOREGOAN (E), MUMBAI-400063 MAHARASHTRA, INDIA
The following specification particularly describes the nature of this invention and the manner in which it is to be performed.

FIELD OF THE INVENTION
The present invention relates to a method of determining bioequivalence of a generic linaclotide drug to the corresponding original linaclotide drug.
BACKGROUND OF THE INVENTION
Development of a new drug (an original drug product) in the pharmaceutical industry requires huge expense and time to perform all stages of development to test the safety and efficacy of the new drug. The company developing a new drug submits a New Drug Application to the regulatory health/medicines authority or agency for an approval to market the new drug. The company developing a new drug (an original drug product) is empowered to recover the expenses as the company owns a patent right with respect to the developed new drug. Once the term of existence of the patent right of the new drug has elapsed, other companies (generic drug makers) are able to manufacture and sell a generic drug product having an active ingredient identical to that of the corresponding original drug product. The examination for approval of a generic drug product is focused on confirmation of equivalence of the generic drug product to the corresponding original drug product. The company developing a generic drug product submits an Abbreviated New Drug Application to the regulatory health/medicines authority or agency for an approval to market the generic drug product. With a demonstration of equivalence, the generic drug company need not perform the expensive and time consuming tests that are required for the original drug product. The equivalence is measured on various specified parameters, one being the bioequivalence. This procedure enables generic drug companies to supply generic drug products at reasonable price.
The linaclotide original product is available as LINZESS® Capsules by Allergan. Allergan filed a citizen petition (Docket No. FDA-2016-P-1962) to FDA requesting that a generic product must demonstrate bioequivalence to LINZESS® Capsules in a clinical endpoint bioequivalence study using appropriate, validated clinical endpoints conducted in patients with Irritable Bowel Syndrome with Constipation ("IBS-C"). Allegan reasoned out by that as linaclotide and MM-419447, are minimally absorbed and that serum levels of linaclotide could only be detected at supratherapeutic doses and serum levels of MM-419447 could not be detected at all in pharmacokinetic studies. They also mentioned the following FDA-approved prescribing information for LINZESS®.

“LINZESS® is minimally absorbed with low systemic availability following oral administration. Concentrations of linaclotide and its active metabolite in plasma are below the limit of quantitation after oral doses of 145 mcg or 290 mcg were administered. Therefore, standard pharmacokinetic parameters such as area under the curve (AUC), maximum concentration (Cmax), and half life (t½) cannot be calculated.”
There were prior attempts to quantify linaclotide or its metabolite by chromatographic techniques. For example, a journal publication (Sree Rama Murthy et al; Acta Chromatographica; DOI: 10.1556/1326.2017.29.2.05) described the quantitative determination of linaclotide and its degradation products by reverse phase high performance liquid chromatography using photodiode array as the detector. However, this prior art method could detect linaclotide at limit of quantitation 7.09 µg/ml and not at picogram levels. Further, this method demonstrated analysis in a pharmaceutical product rather than a biological plasma sample. Another group of analyst reported an analytical method that attempted quantification of linaclotide and its metabolite (Busby et al; J Pharmacol Exp Ther 344:196–206, January 2013). In this prior art method, human plasma sample was analyzed for linaclotide and MM-419447 concentrations using high performance liquid chromatography and mass spectrometry. However, the drawback or limitation is that the method was not sensitive to detect lower levels of linaclotide or MM-419447 in plasma. When a pharmacokinetic study was conducted on healthy human volunteers and quantitation of linaclotide was done using the method, it could not quantify linaclotide and MM-419447 below 3 ng/ml.
The present inventors, have overcome these limitations and have found a method of determining bioequivalence of a generic linaclotide product to the original linaclotide product in plasma samples by measuring the concentration of linaclotide by an analytical method. The limit of quantitation for linaclotide was 1 pg/ml and limit of quantitation for the active metabolite MM-419447 was 2.5 pg/ml. Additionally the method was also able to quantify active metabolite MM-419447, simultaneously. For determining the bioequivalence a scaling approach was employed. In such approaches, the bioequivalence limits of 80-125%, are expanded as a function of within subject variability of the reference product. The method of the present invention also demonstrated a difference of less than ±20% in the point estimate

of the T/R ratio (C and AUC) and thus, a generic linaclotide product may be considered
max
bioequivalent.
SUMMARY OF THE INVENTION
The present invention provides a method of determining bioequivalence of a generic linaclotide product to the corresponding original linaclotide product, the method comprising
a. performing a reference replicated cross-over bioequivalence study,
b. measuring the plasma concentration linaclotide in the plasma sample over 0 to 12
hours using an analytical method that has a limit of quantitation for linaclotide of 1
pg/ml,
A) if within-subject standard deviation of the original linaclotide product product (ΣWR) ≥ 0.294 for BE parameters (AUC0-t, AUC0-inf and Cmax), then bioequivalence standard is established as follows:
i. the 95% upper confidence bound for the linearized criterion [(µT - µR) - 0 Σ2WR ] should be less than or equal to zero for Cmax, AUC0-t, AUC0-oo where, µT & µR are the mean of ln transformed pharmacokinetic parameter (Cmax, AUC0-t, AUC0-∞) obtained from the bioequivalence study for genetic & original products, respectively.
Σ2 WR is the within-subject, within-reference product variance and scaled average BE limit is

Σ 2W0 = 0.25 (regulatory limit) is the cutoff within subject standard deviation
for scaling and
ii. the geometric mean ratio of generic linaclotide product-to-originial
linaclotide product for each of these parameters should be within the interval
80.00 to 125.00 %. B) if Σ2 2WR of the linaclotide original product < 0.294, then two one-sided t-test procedure shall be used and 90% confidence interval of the geometric mean ratio (GMR) Cmax, AUC0-t, AUC0-∞ of the generic linaclotide product and the original linaclotide product should be between 80.00% and 125.00% for ln-transformed data.

The plasma levels of active metabolite MM-419447 may also be measured and the above mentioned analysis performed on to provide the results as a support to the above determination of bioequivalence.
DESCRIPTION OF THE FIGURES
Figure 1 : The figure provides the plasma concentration vs time profile of linaclotide for the
generic linaclotide product and the original linaclotide product (LINZESS®) over a period of
0 to 12 hours.
Figure 2: The figure provides the plasma concentration vs time profile of MM-419447 for the
generic linaclotide product and the original linaclotide product (LINZESS®) over a period of
0 to 12 hours.
Figure 3: The figure depicts the chromatogram when blank plasma sample is run and
detection is carried out by mass spectrometry. The figure shows that no interference was
observed at the retention time of Linaclotide even at the 1.00 pg/mL, which is the lower limit
of quantification (LLOQ) level.
Figure 4: The figure depicts the chromatogram of Linaclotide at its LLOQ. An intense peak
at 6.79 minutes indicates better signal to noise ratio.
Figure 5: The figure depicts the chromatogram when blank plasma sample is run and
detection is carried out by mass spectrometry. The figure depicts that no interference was
observed at the retention time of MM-419447 even at the 2.50 pg/mL which is the lower limit
of quantification (LLOQ) level.
Figure 6: The figure depicts the chromatogram of MM-419447 at it’s lower limit of
quantification (LLOQ) where intense peak at 6.09 minutes of MM-419447 indicating better
signal to noise ratio.
Figure 7: The graph shows linearity over a concentration range of 1.02 to152.69 pg/mL with
correlation coefficients (r) ≥ 0.99 for linaclotide.
Figure 8: The graph shows linearity over a concentration range of 2.54 to 152.37 pg/ml with
correlation coefficients (r) ≥ 0.99 for MM-419447.

DESCRIPTION OF THE INVENTION
The reference replicated crossover bioequivalence study design is the one that allows estimates of within subject variability of reference product which enable the scaling of BE limit of highly variable drug products . It is done by following a three way study design.
A three-way crossover reference replicated design is considered, where the reference product is given twice and test product once for eg TRR, RTR, RRT and scaled average bioequivalence statistical analysis is performed. A washout period adequate to the drug under study (a least 5 half lives) separates each of the three treatment periods. Plasma concentration-time profiles are obtained after each administration, and non-compartmental methods are used to derive summary measures AUC [area under the curve, extent of absorption] and Cmax [rate of absorption]. The Scaled Average Bioequivalence (SABE) regulatory decision rule for demonstrating BE under this design. Linaclotide has the following amino acid sequence and structure: H–Cys1–Cys2–Glu3–Tyr4–Cys5–Cys6–Asn7–Pro8–Ala9–Cys10–Thr11–Gly12–Cys13–Tyr14–OH

MM-419447 is the active metabolite of linaclotide. It has the following structure: H-Cys1-Cys2-Glu3-Tyr4-Cys5-Cys6-Asn7-Pro8-Ala9-Cys10-Thr11-Gly12-Cys13-OH


The present invention provides a method of determining bioequivalence of a generic linaclotide drug to the original linaclotide drug comprising performing a reference replicated cross over bioequivalence study and measuring the plasma concentration of linaclotide and optionally, its active metabolite MM-419447 from the plasma sample using an analytical method that has a limit of quantitation for linaclotide of 1.00 pg/ml the plasma levels of active metabolite MM-419447 may also be measured and the limit of quantitation for the active metabolite MM-419447 of 2.50 pg/ml.
A particular analytical method useful in the quantification of linaclotide and/or its metabolite MM-419447 in plasma is based on a selective, sensitive, accurate and precise liquid chromatography tandem mass spectrometry (LC–MS/MS) method. However, it is possible given the disclosure in the present specification for a person of skill in the art to apply conventional modifications to the method and arrive at similar liquid chromatographic method or apply the principles illustrated by the method and arrive at other analytical methods. It will be thus within skill in the art to arrive at alternative analytical method such as those based on a gas chromatography, thin layer chromatography or high performance thin layer chromatography, gel permeation chromatography or similar techniques.
One skill in the art can select high performance liquid chromatography instruments and columns that are suitable for use in the invention. The chromatographic column typically includes a medium (i.e. a packing material) to facilitate separation of chemical moieties (i.e. fractionation). The medium may include minute particles. The particles include a bonded surface that interacts with the various chemical moieties to facilitate separation of the

chemical moieties. One suitable bonded surface is a hydrophobic bonded surface such as an alkyl bonded surface. Alkyl bonded surfaces may include C-4, C-8, or C-18 bonded alkyl groups, often times, but not isolated to, C-18 bonded groups. The chromatographic column includes an inlet port for receiving a sample and an outlet port for discharging an eluent that includes the fractionated sample. In one embodiment, the sample (or pre-purified sample) is applied to the column at the inlet port, eluted with a solvent or solvent mixture, and discharged at the outlet port. Different solvent modes, or mobile phases, may be selected for eluting the analytes of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytyptic (mixed) mode. During chromatography, the separation of material is affected by variables such as choice of column, eluent (mobile phase), choice of gradient elution and the gradient conditions, temperature etc.
Mass spectrometry (MS) refers to an analytical technique to identify compounds by their mass to charge ratio, MS technology generally includes four components: (1) sample introduction, e.g. HPLC; (2) ionizing the compounds to form charged compounds; (3) separation of the produced ions; and (4) detecting the charged species by monitoring mass to charge ratios. The compound may be ionized and then detected by any suitable means. Mass spectrometry may be performed using a mass spectrometer which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example, ionization of the sample may be performed by electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization, photoionization, electron ionization, fast atom bombardment/liquid secondary ionization, matrix assisted laser desorption ionization, field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. The skilled artisan will understand that the choice of ionization method can be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.
A particular method according to the present invention, used mass spectrometer API-5500 (AB Sciex, Canada) coupled with Dionex UHPLC Ultimate 3000 (Thermo Fisher Scientific, USA). The procedure and its modifications will be understood by a person of skill in the art from the description of the particular process as described herein below:

Preparation of stock solutions for calibration curve for linaclotide:
A mixture of 2 ml plasma (subjected to K2EDTA as an anticoagulant) and 6 ml acetonitrile was vortexed and centrifuged. The supernatant was collected. The supernatant was diluted with water (1:9) to form the diluent, which was used to prepare stock solutions of linaclotide having concentration of 1.02 pg/ml, 2.04 pg/ml, 10.18 pg/ml, 25.45 pg/ml, 45.81 pg/ml, 66.17 pg/ml, 96.70 pg/ml, 122.15 pg/ml, and 152.69 pg/ml. Preparation of stock solutions for calibration curve for MM-419447:
A mixture of 2 ml plasma (subjected to K2EDTA as an anticoagulant) and 6 ml acetonitrile were vortexed and centrifuged. The supernatant was collected. The supernatant was diluted with water (1:9) to form the diluent, which was used to prepare stock solutions of MM-419447 having concentration of 2.54 pg/ml, 5.08 pg/ml, 10.16 pg/ml, 25.40 pg/ml, 45.71 pg/ml, 66.03 pg/ml, 96.50 pg/ml, 121.90 pg/ml, and 152.37 pg/ml. Preparation of internal standard solution of linaclotide:
Internal standard solution is an isotope marked linaclotide (D4 linaclotide). The presence of isotope labeled internal standard in the sample allows for accurate identification and quantification of linaclotide and the metabolite. The same diluent was used to prepare stock solution of 1 mg/ml and working solution os 1.5 ng/ml of D4 linaclotide. Preparation of quality control samples of linclotide and MM-419447 solutions: Quality control sample is a sample with a known quantity of linclotide and MM-419447 that is used to monitor the performance of the bioanalytical method and to assess the integrity and validity of the results of the plasma samples of human volunteers, while determining the plasma levels from the generic linaclotide product. The quality control sample of linaclotide may be prepared at 2.90 pg/ml to 320.48 pg/ml. The quality control sample of MM-419447 may be prepared at 7.50 pg/ml to 324.21 pg/ml.
The concentrations of quality control samples of linaclotide and MM-419447 may for example be as follows:

Table 1: Concentrations of quality control samples of linaclotide and MM-419447

Concentrations of quality control samples Linaclotide (pg/ml) MM-419447 (pg/ml)
Lower range 2.90, 8.61 7.50, 22.39
Medium range 32.05, 68.10 36.47, 68.89
Higher range 125.19 124.62
According to one embodiment of present invention, the method of determninig bioequivalence of a generic linaclotide product to the original linaclotide product includes, performing a reference replicated cross-over bioequivalcne study; by measuring the concentration of linaclotide and optionally, its metabolite MM-419447 in the plasma sample over 0 to 12 hours, using an analytical method that has a limit of quantification for linaclotide of 1 pg/ml. In one of the preferred embodiment, the plasma sample is subjected to a solid phase extraction method. The solid phase extraction method comprises steps of loading the plasma sample onto a non-polar divinylbenzene based neutral polymeric sorbent solid phase extraction cartridge, removing the interfering substances from the cartridge by washing with water, eluting the sample with a solvent, evaporating to dryness, reconstituting and loading the reconstituted solution onto a weak anion exchange cartridge, washing with an acidic solvent system, eluting the sample with a solvent, evaporating to dryness and reconstituting with a solvent. The reconstituted sample is then subjected to a High Performance Liquid chromatography by injecting the reconstituted sample into an analytical column made up of reverse phase C-18 silica particles, having an average particle size of 1.9 microns, using a mobile phase made up a mixture of organic solvent and aqueous buffer having pH in the range of 2 to 4 and detecting and quantifying the linaclotide and optionally, MM-419447 using mass spectrometry.
One specific method is descbribed as follows:
Preparation of analyte samples (Linaclotide and MM-419447):
In this, linaclotide and/or MM-419447 were extracted from the plasma sample by first
passing the plasma sample through two solid phase extraction cartridges one after another.
However, instead of a cartridge, it is possible to use a polymeric fibre membrane, a glass
fibre membrane, a multi-well plate or microelution plates. In one preferred embodiment, the
plasma sample is passed into first non-polar polymeric sorbent cartridge followed by an ion
exchange resin cartridge, for eg. anionic exchange resin cartridge. The non-polar polymeric
sorbent cartridge is selected from a sorbent cartridge with polymeric backbone selected from

poly(styrene divinylbenzene), copolymers of styrene, copolymers of divinylbenzene, functionalized styrenes, functionalized heterocycles and combinations thereof. In one of the examples, the non-polar divinylbenzene based neutral polymeric sorbent cartridge was chosen which provided performance enhancements due to an unique polymeric architecture with a non-retentive, hydroxylated, amide-free surface and a non-polar poly(styrene/divinylbenzene) core. This particular type of polymeric sorbent is water wettable wherein hydroxylated exterior allows excellent flow of biofluid samples. A gradient of polarity on the polymer surface shunts analytes to the more hydrophobic center of the polymer bead where they are retained prior to washing and elution steps. Binding of proteins and lipids on the polymer surface is minimized, resulting in cleaner samples and reduced ion suppression. This resulted in higher recoveries which translates into better sensitivity. The cartridge packing has nominal pore size of 120 Å whereas the particle size of the sorbent is 45 µm with specific surface area 450 m2/g and is stable over a pH range of 1 to 14. The second ion exchange resin cartridge was selected from sorbent having backbone of strong cation exchange fuctionalized polymeric sorbent, weak cation exchange fuctionalized polymeric sorbent, strong anion exchange fuctionalized polymeric sorbent and weak anion exchange cartridge. For example, the weak anion exchange functionalized polymeric sorbent was used comprising sorbent mass of 30mg, 60mg,100mg, 200mg and 500mg that allows complete retention of acidic compounds with pKa value less than 5. Di-amino functional group of the sorbent is highly selective for charged acidic compounds and allows for 100% organic washes, increasing sensitivity and method reproducibility. The cartridge has pore size of 85 Å and particle size of sorbent 33µm.
In a specific example, the plasma sample was subjected to K2EDTA as an anticoagulant and it was loaded into pre equilibrated non-polar divinylbenzene based neutral polymeric sorbent cartridge. The process is then followed by washing with water to remove interfering substances. Further, the samples were eluted from the column with 500 uL of methanol in acetonitrile (50% v/v). Further, the sample was eluted with ethanol in water (90% v/v) to achieve optimum recovery of linaclotide and MM-419447 present in the analyte samples. After elution, sample was collected and evaporated at 50°C to dryness and then reconstituted with 150uL of 0.1 % acetic acid in methanol: water; 300 µL of 0.1% ortho phosphoric acid and 300 µL of water. The reconstituted sample was further loaded into pre equilibrated weak anion exchange cartridge. This cartridge was then washed with 1% formic acid followed by methanol to remove any interfering substances. The sample was then eluted from the

cartridge using 5% v/v ammonia solution in methanol in ethanol (50% v/v). The eluted sample was evaporated at 50°C to dryness. The sample was then reconstituted with 150uL of 0.1 % Acetic acid in methanol: water; and transferred the content into HPLC vial for analysis.
Detection and quantification of linaclotide and its metabolite MM-419447
A gradient chromatographic separation using a reverse phase column was performed. According to the present invention, any chromatographic column that can provide good resolution between the linaclotide and MM-419447 peaks, i.e which can result into an efficient separation of both linaclotide and MM-419447, free from interference at the retention time of each other, free from matrix effect and better S/N ratio at LLOQ level, can be used. When the method employs liquid chromatography mass spectrometry as the technique to quantify linaclotide and MM-419447, the inventors found that a very good, acceptable resolution was achieved. A signal to noise ratio of more than 5 was obtained, for e.g. 5.9. The resolution parameters such as signal to noise ratio for such techniques is described in (EMA/434565/2010; Committee for Medicinal Products for Human Use (CHMP).
In one example, a column with a surface area of 350 m2/g, pore size of 10 nm and pore
volume of 0.85 ml/g with octadecyl groups of bonded phase of the chromatographic column
with highly inert packing provided less tailing of peaks and provided a acceptable resolution
between the two peaks.
In one specific example, the method used the following parameters.
Column details: C-18 reverse phase column, with particle size of silica 1.9 µm
Mobile phase A: methanol
Mobile phase B: 0.25 % acetic acid in water
Mobile phase flow rate: 0.150 ml/min
Injection volume: 30 µL
Table 2: Mobile phase gradient elution details

Gradient Time Flow rate ml/min % A % B

0 0.150 20 80

8.0 0.150 100 0

8.1 to 10.0 0.150 80 80

Linaclotide was detected at 6.77 minutes and MM-419447 was detected at 6.07 minutes and linaclotide D4, was detected at 6.75 minutes respectively. Stock solutions of various concentrations of each were run and area response was measured. Mass spectrometry detection was carried out in positive electrospray ionization mode with multiple reactions monitoring (MRM) scan. (M+2)/2 fragment ions were used to quantify linaclotide and MM-419447. The selective reaction monitoring transitions m/z 764.0 to 182.1 m/z were used for the determination of linaclotide and for MM-419447, the m/z was 682.4 to 136.1. For D4 linaclotide, the monitoring transitions m/z 766.4 0 to 182.1 m/z were used.
The calibration curves were obtained by recording the peak area ratios vs. nominal concentration. The calibration curves for each, was linear as determined by weighted (1/x2) linear regression. Linear calibration curve was obtained for linaclotide at a concentration range of 1.02-152.69 pg/mL, whereas for MM-419447 the calibration curve was linear at the concentration range of 2.54-152.37 pg/mL with correlation coefficients (r) ≥ 0.99. (See Figure 7 and Figure 8). The method of the present invention was found to be very sensitive as quantification of linaclotide and MM-419447 at very low concentrations till pictogram levels could be achieved. The method was also very accurate. This is evident because when linaclotide was analyzed at a lower limit of quantification of linaclotide of 1.02 pg/ml, the analytical method according to the present invention measured the concentration in the range of 0.82 to 1.22 picogram/ml. This was true for all the intermediate as well as higher concentration ranges, for eg. for a concentration of 2.90 picogram/ml, the method quantified it in a very narrow range of 2.47 to 3.34, indicating a minimal deviation. This was applicable to higher concentrations as well, for eg. for a 152.69 pg/ml, the method quantified in a very narrow range of 120.79 to 175. 59, indicating a minimal deviation. Thus, the analytical method is indeed not only sensitive but accurate, with less co-efficient of variation or low standard deviation, at all the concentrations at which the concentration responses were linear. This was applicable to the active metabolite as well. In case of the metabolite, lower limit of quantification was 2.54 pg/ml. The analytical method according to the present invention measured the concentration in the range of 2.03 to 3.05 picogram/ml. All the intermediate as well as higher concentration ranges, for eg. for a concentration of 7.50 picogram/ml, the method quantified it in a very narrow range of 6.38 to 8.69, indicating a minimal deviation.
Mean extraction recoveries were approximately 60% for both linaclotide and MM-419447 where analyte samples were tested. Intra- and inter-run mean % accuracy were between 85–

115% except lower limit of quantification where it was between 80–120% and % imprecision was ≤ 15% except lower limit of quantification where it was ≤ 20%.
The above described method was applied to determine bioequivalence of the generic linaclotide product (A) to original linaclotide product (B). The below protocol was followed: This was a randomized, open label, two treatment, three period, three sequence, single dose, reference-replicated crossover, bioequivalence study of hard gelatin capsules of generic linaclotide product having 290 mcg of linaclotide filled in hard geltin caspules and original linaclotide product, availabile under the brand name of Linzess 290 mcg Capsules. Totally, 42 healthy adult subjects were made to take either generic or original linaclotide product under fasting condition. Prior to study commencement, subjects were randomly assigned to a treatment sequence in accordance with the randomization scheme generated by biostatistician.
Single oral doses were separated by washout period of six (06) days (between period-I and II) and seven (07) days (between period-II and III).
Treatments administered
Test (A): Single oral dose 290 mcg of generic linaclotide product capsule (3 x 290 mcg
Capsule at a time or one after another) was administered with about 240 mL of water at
ambient temperature, in the morning according to the randomization schedule.
Reference (B): Single oral dose of original linaclotide product having brand name of Linzess
(Linaclotide 290 mcg Capsule (3 x 290 mcg Capsule at a time or one after another)) was
administered with about 240 mL of water at ambient temperature, in the morning according
to the randomization schedule.
Two treatments and three sequences were used in this study. In the sequence 1, treatment A
was given in period I and treatment B was given in period II and III. In sequence 2, treatment
B was given in period I and III, whereas treatment A was given in period II. In sequence 3,
treatment B was given in period I and II, followed by treatment A in period III.
Drug administration and Compliance
Generic linaclotide product and original linclotide product were directly placed in subject’s mouth Subjects then swirled the mouth with dosing water and gulped the remaining dosing water. Subjects were dosed as specified in the protocol and subsequently fasted for 4 hours

post dose in each period. Dosing was performed under direct supervision of study physician or designated study personnel. Compliance was assessed by conducting a thorough examination of oral cavity by trained study personnel after dosing in each study period to ensure that the capsules were swallowed as a whole without chewing or biting.
Primary Pharmacokinetic variables
The pharmacokinetic parameters Cmax, AUC0-t and AUC0-inf were taken as primary
pharmacokinetic variables for establishing the bioequivalence.
Cmax : Maximum measured plasma concentration over the time span specified
AUC0-t : The area under the plasma concentration versus time curve, from time 0 to the last
measurable concentration, where t = time of last identifiable concentration.
AUC0-inf : The area under the plasma concentration versus time curve from time 0 to infinity.
It is calculated by adding Ct/Kel to AUC0-t were Ct is the last quantifiable concentration and
Kel is the elimination rate constant.
Drug concentration measurements
A total 19 blood samples were drawn into vacutainers containing K2EDTA as an anticoagulant, prior to drug administration pre-dose (14 mL) and at 0.167, 0.250, 0.333, 0.417, 0.500, 0.750, 1.000, 1.333, 1.667, 2.000, 2.500, 3.000, 4.000, 5.500, 7.000, 8.000, 10.000 and 12.000 hours post dosing (1 x 7 mL each) in each study period. The pre-dose and post-dose samples were collected by direct vein puncture. The pre-dose samples were collected within a period of 1 hour prior to schedule dosing and the post dose samples were obtained as per their scheduled time or within the specified window period of 2 minutes during housing. The plasma samples were analysed for linaclotide and its active metabolite MM-419447. The data for linaclotide establishes the bioequivalence according to criteria as claimed whereas data for MM-419447 was a supportive data. Figure 1 provides the plasma concentration vs time profile of linaclotide for the generic linaclotide product and the original linaclotide product (LINZESS®) over a period of 0 to 12 hours. Figure 2 provides the plasma concentration vs time profile of MM-419447 for the generic linaclotide product and the original linaclotide product (LINZESS®) over a period of 0 to 12 hours.
Statistical Methods Planned in the protocol and Determination of Sample Size Statistical Analysis Plan

A validated LC-MS/MS bio analytical method developed for the quantification of Linaclotide and its active metabolite MM-419447 in plasma. In accordance with study protocol this method was employed on samples from all subjects who had completed at least two periods of the study but analysis of samples was not done for the particular period in which subject dropped out / withdrawn.
Pharmacokinetic Analysis
Following pharmacokinetic parameters were calculated for linaclotide and its active metabolite MM-419447.
Cmax, AUC0-t, AUC0-inf, %AUC extrapolation, Tmax, Kel, t1/2, TLIN and LQCT
Pharmacokinetic parameters were calculated using Phoenix® Win-Nonlin® (Version 6.4) using non-compartmental analyses. The arithmetic mean, standard deviation (SD), coefficient of variation (CV %) and range (min. and max.) are calculated for plasma concentrations of linaclotide and its active metabolite MM-419447. for each sampling time and treatment. The arithmetic mean, SD, CV (%), median, geometric mean and range (min. and max.) were calculated for the Cmax (pg/mL), AUC0-t (pg.h/mL), AUC0-inf (pgh/mL), %AUC Extrapolation, Tmax (h), t1/2 (h), Kel (1/h), TLIN (h) and LQCT (h).
Statistical Analysis
Statistical analyses were performed on individual pharmacokinetic parameters obtained for linaclotide and its active metabolite MM-419447, using the SAS package (SAS Institute Inc., USA, Version 9.4). Analysis of variance was performed on both Un-transformed and Ln-transformed data of AUC0-t, AUC0-inf and Cmax. All ANOVAs were performed with the SAS (release 9.4 for Windows).
In accordance with the study protocol, following bioequivalence criteria were followed:
1) If within-subject standard deviation of the reference product (GWR) > 0.294 for BE parameters (AUC0-t, AUC0-inf and Cmax), then bioequivalence standard would be established as follows:
i. The 95% upper confidence bound for the linearized criterion [(µT - µR) - 9 O WR ] should be less than or equal to zero for Cmax, AUC0-t, AUC0-oo

Where, µT & µR are the mean of ln transformed pharmacokinetic parameter (Cmax, AUC0-t, AUC0-∞obtained from the bioequivalence study for generic linaclotide product & original linaclotide product, respectively.
σ 2WR is the within-subject, within-reference product variance and scaled average BE limit is

σ 2W0 = 0.25 (regulatory limit) is the cutoff within subject standard deviation for scaling and
ii. The geometric mean ratio of generic linaclotide product -to- original linaclotide product for each of these parameters should be within the interval 80.00 to 125.00 %.
2) If σ 2WR of the reference product < 0.294, then two one-sided t-test procedure shall be used and 90% confidence interval of the geometric mean ratio (GMR) Cmax, AUC0-t, AUC0-∞ of the generic linacotide product and original linaclotide product should be between 80.00% and 125.00% for ln-transformed data.
Determination of within subject CV% for reference product:
An intermediate analysis using PROC GLM procedure was performed in order to calculate within-subject standard deviation of the reference product (σWR). Statistical output by intermediate analysis using GLM procedure for linaclotide and for active metabolite MM-419447.
Linaclotide
Since within-subject standard deviation of reference product (σWR) found more than 0.294 for AUC0-t (i.e., 0.428; N=36), AUC0-inf (i.e., 0.399; N=36) and Cmax (i.e., 0.418; N=36) by intermediate analysis using GLM procedure, BE Criteria 1 was followed for above mentioned parameters.
Active metabolite MM-419447
Since within-subject standard deviation of reference product (σWR) found more than 0.294 for AUC0-t (i.e., 0.517; N=36), AUC0-inf (i.e., 0.453; N=23) and Cmax (i.e., 0.494; N=36) by intermediate analysis using GLM procedure, BE Criteria 1 was followed for above mentioned parameters.

Assessment of Bioequivalence: Linaclotide and optionally its active metabolite MM-419447
For AUC0-t, AUC0-inf and Cmax, ANOVAs were performed twice with the SAS (release 9.4 for Windows) General Linear Model Procedure. The first ANOVA was performed on difference between the individual Ln-transformed AUC0-t, AUC0-inf and Cmax data of generic linaclotide product and original linaclotide products (where the reference data is average of the replicates of the reference drug) including sequence effect as a fixed effect. The second ANOVA model was performed on difference between the individual Ln-transformed data of AUC0-t, AUC0-inf and Cmax for reference products (which was administered twice) including sequence effect as a fixed effect.
The Ln-transformed pharmacokinetic parameters (AUC0-t, AUC0-inf and Cmax) were evaluated for scaled average bioequivalence (SABE) using a linearized criterion for linaclotide and active metabolite MM-419447. The SABE and the linearized equations used were:
within-subject, within-reference product variance).
The 95% upper confidence bound for linearized statistic (ŋ) was estimated from the Ln-transformed AUC0-t, AUC0-inf and Cmax data for linaclotide and active metabolite MM-419447. Where linearized statistic (ŋ) was modified as (µT - µR) - S.E - θ*σ 2 WR to account for bias correction on the squared difference between the test and reference means, as per the new recommendation from FDA for SABE in the Progesterone guideline where S.E is the standard error obtained from first ANOVA. The Reference coefficient of variation was estimated from the Reference variance term σ 2 WR) of the Ln-transformed (loge) results as: 100% * SQRT (es -1) (where s WR is the unbiased estimate of σ 2 WR obtained from the respective ANOVA models for AUC0-t, AUC0-inf and Cmax).
For the generic linaclotide product and original linaclotide products to be declared as bioequivalent, the 95% upper confidence bound of linearized statistic (ŋ) should be less than or equal to zero and the GMR (Geometric Means ratio) values must fall within the interval 80.00% to 125.00% as another criteria of scaled average bioequivalence.
For Active metabolite MM-419447, above mentioned statistical analysis was carried to provide the results as a supportive data but not included in bioequivalence criteria.

We claim:
1. A method of determining bioequivalence of a generic linaclotide product to the original linaclotide product, the method comprising
a. performing a reference replicated cross-over bioequivalence study,
b. measuring the concentration of linaclotide in the plasma sample over 0 to 12
hours using an analytical method that has a limit of quantitation for linaclotide of
1 pg/ml,
A) if within-subject standard deviation of the original linaclotide product product (ΣWR) ≥ 0.294 for BE parameters (AUC0-t, AUC0-inf and Cmax), then bioequivalence standard is established as follows:
i. the 95% upper confidence bound for the linearized criterion [(µT - µR) - 0 Σ2WR ] should be less than or equal to zero for Cmax, AUC0-t, AUC0-∞ where, µT & µR are the mean of ln transformed pharmacokinetic parameter (Cmax, AUC0-t, AUC0-obtained from the bioequivalence study for generic linaclotide product & original linaclotide product, respectively, Σ2 WR is the within-subject, within-reference product variance and scaled average BE limit is

σ 2W0 = 0.25 (regulatory limit) is the cutoff within subject standard deviation for scaling and
ii. the geometric mean ratio of generic linaclotide product-to-original linaclotide product for each of these parameters should be within the interval 80.00 to 125.00 %;
B) if σ 2WR of the original linaclotide product < 0.294, then two one-sided t-test procedure shall be used and 90% confidence interval of the geometric mean ratio (GMR) Cmax, AUC0-t, AUC0-∞ of the generic linaclotide product and original linaclotide product should be between 80.00% and 125.00% for ln-transformed data. 2. A method as claimed in claim 1, wherein the method measures the concentration of MM-419447 in the plasma sample.

3. A method as claimed in claim 1 of 2, wherein the method comprises extracting linaclotide and/or MM-419447 from the plasma sample by solid phase extraction method using a non-polar divinylbenzene based neutral polymeric sorbent solid phase extraction cartridge and a weak anion exchange cartridge.
4. A method as as claimed in claim 3 wherein the solid phase extraction method comprises steps of loading the plasma sample onto a non-polar divinylbenzene based neutral polymeric sorbent solid phase extraction cartridge, removing the interfering substances from the cartridge by washing with water, eluting the sample with a solvent, evaporating to dryness, reconstituting and loading the reconstituted solution onto a weak anion exchange cartridge, washing with an acidic solvent system, eluting the sample with a solvent, evaporating to dryness and reconstituting with a solvent.
5. A method as as claimed in claim 4, the method comprising subjecting the reconstituted sample of claim 4 to High Performance Liquid chromatography by injecting the reconstituted sample into an analytical column made up of reverse phase C-18 silica particles, having an average particle size of 1.9 microns, using a mobile phase made up a mixture of organic solvent and aqueous buffer having pH in the range of 2 to 4 and detecting and quantifying the linaclotide and/or MM-419447 using mass spectrometry.