Study Treatments
Formulation of Powder in Oily Suspension
OS (TPE™; Chiasma, Jerusalem, Israel) was prepared using sodium caprylate as illustrated in Fig. 1 (16,17). Briefly, sodium caprylate (Merck, Darmstadt, Germany) and polyvinyl pyrrolidone (PVP; Kollidon 12, BASF, Burgbernheim, Germany) with or without octreotide acetate (Bachem, Torrence, CA) or fluorescein isothiocyanate (FITC)-labeled dextran (Sigma, St. Louis, MO) were solubilized in water, milled (particle size ranging 90 – 100 μm), lyophilized, and suspended in lipophilic medium consisting of polysorbate 80 (PS 80; Croda, UK), glyceryl monocaprylate (GMC; Abitec, Janesville, WI) and glyceryl tricaprylate (GTC; Abitec). The formulated OS was subsequently filled into hard-shell gelatin capsules (Capsugel, Bornem, Belgium) that were banded with prewarmed gelatin and 1% Twin 20 solution using a Dott Bonapace & Co. (Milan, Italy) semi-automatic banding machine, and dried at room temperature. The OS capsules were next enteric coated with a 20% aqueous suspension of Acryl-EZE® (# 93018509; Colorcon, Dartford, UK) using a perforated coating pan system (Compu-Lab, Thomas Engineering Inc., Hoffman Estate, IL) to reach a 90 mg capsule weight gain. The capsules were stored refrigerated (2 to 8°C) prior to their use.
The octreotide/OS capsules were tested for physical appearance, potency (assay), dissolution, microbiological and water content tests before their release and during the chronic toxicity study under refrigerated (2 to 8°C) and controlled room temperature (25°C and 60% relative humidity) conditions. The capsules appearance, white matt coated capsule, was maintained throughout the entire stability period. The octreotide content in the octreotide/OS capsules was analyzed by HPLC using Waters Alliance 2695 XE (Waters Corp., Milford, MA) equipped with Waters Photodiode Array Detector 2996 at λ = 210 nm. Briefly, the capsules were dissolved in 65 mM ammonium sulfate solution and analyzed by HPLC, using a C18 column (YMC Co., Ltd. Kyoto, Japan, 4.6 × 150 mm, 3 μm) with the following mobile phase: water (A), acetonitrile (B), 0.1% phosphoric acid and 20 mM sodium-1-decane sulfonate (C). The reported values were based on the signal area at the appropriate retention time. The octreotide content in the refrigerated octreotide/OS capsules confirmed with the drug specifications over the 9-month period. In addition, the drug release profiles from the enteric coated octreotide/OS capsules were carried out using a USP-II apparatus (Caleva 8ST dissolution apparatus, GB Caleva, Dorset, England) with a two-stage dissolution method according to USP <711> and Ph. Eur. 2.9.3. The tests were conducted in hexaplicates, in 900 ml dissolution medium maintained at 37 ± 0.5°C. A paddle speed of 50 rpm was employed. The tests consist of a 2-h acid stage dissolution in 0.1 N HCl (pH 1.2) with two sampling time points, and a 1-h buffer stage dissolution in pH 6.8 phosphate buffer with five sampling time points. The medium was changed by the analyst from the acid to the buffer after the first stage had been completed. The dissolution aliquots were withdrawn from the dissolution bath at the indicated time points and analyzed using the HPLC method for octreotide quantification. Samples were analyzed with a Phenomenex Luna C18 column (Phenomenex Co., Torrance, CA, 3.0 × 150 mm, 5 μm) at a flow rate of 0.6 mL/min and mobile phase containing acetonitrile-water-trifluoroacetic acid using Waters Alliance 2695 XE equipped with Waters Photodiode Array Detector 2996 at λ = 220 nm. Again, comparable dissolution profiles (Fig. 2) were obtained for the refrigerated octreotide/OS capsules during the 9-month period.
Animal Treatments
Enteric-coated capsules containing octreotide (20 mg)/OS or olive oil were administrated daily per os to fasted monkeys for 9 months, and swallowed with 10 mL water. In addition, a 1 mL ampule containing octreotide acetate at a concentration of 0.1 mg/mL (Sandostatin®, Novartis Pharmaceuticals, East Hanover, NJ) was administered by daily subcutaneous (SC) injection to another monkeys group. Oral administration of OS in rodent studies was limited due to the lack of properly enteric coated mini-gelatin capsules. Thus, the OS, containing fluorescent marker molecules (see below) or octreotide was enterally injected via implanted cannulas.
Animals
Monkeys
Captive-bred, colony-raised, naive male and female Cynomolgus monkeys (Macaca fasicularis) were obtained from Covance Research Products, Inc. (Denver, PA) and acclimated for 9 days prior to treatment. In compliance with Good Laboratory Practices (GLPs), monkeys were housed individually in suspended stainless-steel cages. Room temperature was controlled to 18 – 29°C, humidity to 30% – 70% and cycled lighting (12 h of light daily). Prior to initiation of the study, the monkeys were in good health, and free of internal parasites and tuberculosis. Certified primate diet (Lab Diet # 5408 Primate Diet, PMI Nutrition International, Inc, Brentwood, MO) was provided twice daily (unless otherwise specified) on a feeding regimen of six to eight biscuits, supplemented by fruits (up to 5 per day). Water was provided ad libitum. Analysis for specific pathogenic microorganisms and contaminants showed no significant findings.
The study was carried out in an Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited facility and in compliance with the Animal Welfare Act regulations (9 CFR 1). All aspects of this study were conducted in accordance with the Environmental Protection Agency and FDA based upon Good Laboratory Practice Regulations, 40 CFR 792.
Rats
Intestinal permeability experiments were performed in vivo using adult male albino Sprague–Dawley (SD) rats (300–400 g bodyweight), obtained from Harlan (Jerusalem, Israel) and acclimatized for 1 week in controlled temperature (22 ± 3°C) and 12-h light and dark cycles. Animals were housed individually, allowed access to rat chow (2018SC Harlan Teklad, Madison, WI) and tap water ad libitum. Animals were fasted for 16 to 18 h overnight before the start of the experiment, between 0700 and 0900 h. Study protocols and procedures were approved by the National Council for Animal Experimentation, Ministry of Health, Jerusalem, Israel.
Chronic Toxicity Study in Cynomolgus Monkeys
Tissue Sampling and Preparation
At study initiation, monkeys weighing 3.34– 6.24 kg were randomly assigned (5/sex/group) to daily administration of single octreotide/OS capsule or control olive oil (negative control) by intragastric intubation or to SC injection of octreotide acetate (positive control) for 9 months. During the dosing period, animals were fasted for 8 to 10 h prior to dosing, and the first feeding was given 2 h after dosing. Monkeys were observed twice daily for mortality, morbidity, and clinical signs of adverse health effects and qualitative food consumption. Weekly examinations were performed on each animal before drug administration and before terminal sacrifice. Pre-dose body weights were recorded (week −1) and weekly thereafter. Blood samples (~5 ml) were collected for hematology and serum chemistry from the femoral vein before drug administration and from surviving animals prior to the terminal necropsy.
A complete ophthalmic examination was performed on each monkey by a Diplomat of the American College of Veterinary Ophthalmologist veterinarian before animal selection/group assignment (pretest) and again, prior to study termination.
Electrocardiograms (ECGs) were obtained twice pre-study and were analyzed by a veterinary cardiologist for the presence of significant abnormalities. ECGs were also obtained following the 1st dose and at the 3, 6, and 9 months of dosing. Standard ECGs (10 Lead) were recorded at 50 mm/s. Using Lead II (or another appropriate lead), the RR, PR, and QT intervals, and QRS duration were measured and heart rate was determined. The QT interval was calculated using Bazett’s Rule.
On the 270th day, the monkeys were fasted overnight, weighed on the following morning, and bled for required tests; then exsanguinated, necropsied, and examined for gross alterations. Animals were euthanized with intravenous ketamine sodium pentobarbital solution. Exsanguination was achieved by severing the femoral vessels.
Toxicokinetics
On Day 1 and after 3, 6 and 9 months, whole-blood samples (3.0 mL) were collected at routine intervals after completion of oral octreotide dosing. Plasma octreotide levels were determined by a validated liquid chromatographic spectrometric method (PPD Laboratories, Richmond, VA) as described elsewhere (18). The maximum plasma concentration (Cmax) and time to Cmax (Tmax) were derived from the data. The area under the curve (AUC) from zero to the last sample with a concentration ≥LOQ [AUC(0-t)] was calculated using the linear trapezoidal method. Analysis of plasma octreotide concentration vs. time was performed using SAS® for Windows® Version 9.3. The primary pharmacokinetic end point variables (Cmax and AUC0-t) were compared using analysis of variance (ANOVA) with treatment time (1, 180, 270-d) as a within-subject (repeated measures)-factor and sex as a between-subject factor.
In Vivo Intestinal Permeability Assay
Intestinal paracellular permeability was determined by measuring the appearance, in the circulation, of a fluorescence marker, 4.4 kDa FITC-labeled dextran (FD4; Sigma). The assay of intestinal paracellular permeability was slightly modified from previously described methods (19–21). Briefly, implantation of jugular venous and intestinal catheters was carried out in SD rats under anesthesia, induced by intraperitoneal (i.p.) injection of ketamine hydrochloride (Kepro, Deventer, Holland) at 95 mg/kg bodyweight and xylazine hydrochloride (Sedaxylan; Eurovet Animal Health BV, Bladel, Holland) at 9.5 mg/kg bodyweight. Implantation of the catheters was carried out 5-7-days prior to the experiments. A thin heparinized polyurethane-based elastomer catheter (Micro-Renathane® MRE 025, 0.305 mm × 0.635 mm; Braintree Scientific Inc., Braintree, MA) was inserted via a 1-cm longitudinal skin incision, ventrally along the midline of the neck, into the right jugular vein. The proximal jejunum was localized immediately caudal to the ligamentum duodenocolicum, via a 2-cm ventral midline incision, and a silastic tubing (0.762 mm × 1.651 mm; Degania Silicone Co. Ltd, Degania Bet, Israel) tippet was inserted via enterotomy to the jejunum. Further experiments involved implanting an additional thin silastic tubing (0.500 mm × 1.000 mm; Degania Silicone) into the jejunum, adjacent to the 0.762 mm cannula. In a single series of experiments, a silastic catheter was inserted 2 cm distal to the pylorus (duodenum), 5 cm proximal to the cecum (ileum) or 1 cm distal to the cecum (colon). Both jugular and intestinal catheters were subcutaneously tunneled, exteriorized behind the animal shoulder blades and glued (histoacryl glue; B. Braun, Melsungen, Germany). Catheters were subsequently filled with heparinized saline and sealed. At the end of the surgery, all skin openings were closed with 0.5 silk sutures and stainless-steel wound clips.
For intestinal permeability experiments, 0.3 mL saline or OS containing 1.65 mg of FD4 was injected via the intestinal cannula into the lumen of conscious non-restrained rats. OS effect on permeability was also tested by administration of OS and fluorescent dextran using two jejunal cannulas, in which these marker molecule solutions in saline served as a “bystander” to the OS permeation activity. Additionally, in a single series of experiments, FITC-labeled dextrans of different molecular weights (average 10, 20, 40 and 70 kDa; Sigma) at an equivalent dose served as the permeability markers. Baseline blood samples (500 μL) were collected from the indwelling jugular catheter pre-dose, and at the indicated times. Blood was centrifuged at 4°C, 3,000×g for 10 min, and the plasma was separated for the analysis of FITC-dextran concentration. Plasma was diluted 1:1 with 5% sodium bicarbonate solution (Biological-Industries, Beit Haemek, Israel) in duplicate, and fluorescence intensity of the diluted plasma measured using a fluoro-spectrophotometer plate reader (Victor Wallac 1420 Multi-label; Perkin Elmer, Wellesley, MA) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Plasma FITC-dextran levels are presented in arbitrary fluorescent units (AU).
Immunohistochemistry
Experiments were conducted in anesthetized SD rats, using a ketamine-xylazine mixture administered i.p., and animals were kept on warming pads (Bair Hugger®, Augustine Medical Inc, Eden Prairie, MN) to maintain body temperature at 38°C. The peritoneum was opened by midline incision and the proximal jejunum identified. After washing the intestines with 2 mL pre-warmed (37°C) saline solution, 0.3 mL saline solution or OS was injected 3 times (0, 10- and 20-min) into the animal lumen. At the indicated time-point, approximately 1-cm of the intestinal section was removed and washed with ice-cold phosphate-buffered saline (PBS).
Frozen sections of jejunal tissues were fixed with 4% paraformaldehyde in PBS for 2 h at 4°C, and then washed thrice with ice-cold PBS for 5 min. Next, jejunal sections were incubated with 30% (wt/wt) sucrose overnight in PBS at 4°C. Jejunal tissues were embedded in OCT cryostat freezing medium compound (Electron Microscopy Sciences, Hatfield, PA), snap-frozen in liquid nitrogen, cryostat sectioned (−80°C) at 14 μm, then mounted on Superfrost/Plus (Menzel Glaser GMBH, Braunschweig, Germany) microscope pre-coated slides.
Sections were incubated with monoclonal rabbit antibody against zonula occludens-1 (ZO-1; Zymed Laboratories Inc., San Francisco, CA) and against claudin-3 (Invitrogen Corporation, Carlsbad, CA) diluted at 1:100 in PBS/0.1% Tween-20 (PTW) for 72 h at 4°C. After washing thrice in blocking solution [Tween 20 (2%), heat inactivated goat serum (10%; Biological Industries, Kibbutz Beth Haemek, Israel) and PBS] for 5 min, sections were incubated with secondary Alexa Fluor 555-conjugated goat anti-rabbit IgG antibody (Invitrogen) at 1:200 for 1 h at 4°C. After washing thrice in blocking solution, sections were mounted on cover slides and images obtained using a laser-scanning confocal microscope (Zeiss LSM 510/Axiovert 100 M, Jena, Germany) and oil immersion objective (×40; NA 1.25).
Rat jejunal sections were also treated with sulfo-NHS-LC-biotin (Pierce, Rockford, IL) mixed with saline or OS and then washed, frozen and sectioned in a comparable procedure. Non-specific binding sites were blocked with blocking solution and 1 μg/mL non-labeled streptavidin (Zymed) was added for 1 h at room temperature. Sections were incubated for 30-min with 10 μg/mL of AlexaFluor-633 conjugated streptavidin (Invitrogen) in blocking solution. After washing thrice in blocking solution, sections were again incubated with the blocking solution and 1 μg/mL non-labeled streptavidin. Sections were then stained for actin by incubation with Alexa Fluor 555 Phalloidin (Invitrogen) diluted at 1:100 in the blocking solution for 30-min at 37°C. After washing thrice in blocking solution, sections were mounted on cover slides and images obtained using confocal microscopy.
Immunoassays
Octreotide and growth hormone (GH) concentrations were assayed in rat plasma samples, following intra-jejunal dosing of 0.3 mL saline or octreotide/OS to cannulated rats. Octreotide acetate in saline was also injected subcutaneously to this animal model.
The octreotide immunoassay is based on a single-step, extraction-free, simultaneous assay procedure (Peninsula Laboratories, Division of Bachem, San Carlos, CA) designed to quantify octreotide concentrations. Briefly, octreotide standards (0.05–6.25 ng/mL) and rat plasma samples were diluted 1:10 with PBS, followed by addition of octreotide anti-rabbit antiserum antibody, biotinylated octreotide tracer, and assay buffer. Samples were washed and incubated with streptavidin–HRP. Optical density was read at a dual absorbance of 450 nm and 630 nm using an Enzyme-Linked Immunoassay (ELISA) reader (Multiskan EX Labsystems; Thermo Electron Corporation, Milford, MA) after addition of TMB solution and acidic stopping solution.
Plasma GH was measured by ELISA kit according to the manufacturer’s instructions (Diagnostic Systems Laboratories, Webster, TX).
Data Analysis
Results were expressed as mean ± SE. The area under plasma concentration–time profile measured from time zero to the last measurable plasma concentration was calculated according to the linear trapezoidal rule. Relative enteral bioavailability (rBA) following intra-jejunal administration of the OS formulation to rats was calculated compared with SC injection as follows:
$$ rBA\%=\left({\mathrm{AUC}}_{\mathrm{jejunum}}/{\mathrm{AUC}}_{\mathrm{SC}}\right)\times \left({\mathrm{Dose}}_{\mathrm{SC}}/{\mathrm{Dose}}_{\mathrm{jejunum}}\right)\times 100 $$
Data were analyzed using ANOVA and a post hoc comparison between groups was conducted using the Student’s t-test (GraphPad, Inplot, San Diego, CA). A significance level of P < 0.05 was used for all statistical analyses.