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Oral Delivery of Antisense Oligonucleotides in Man
Oral Delivery of Antisense Oligonucleotides in Man LLOYD G. TILLMAN, RICHARD S. GEARY, GREGORY E. HARDEE ISIS Pharmaceuticals, Inc., 1896 Rutherford Road, Carlsbad, California 92008
Received 18 October 2006; revised 20 December 2006; accepted 17 January 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21084
ABSTRACT: Treatment of systemic disease with phosphorothioate antisense oligonucleotides (PS ASOs) has been accomplished using local or parenteral routes of administration to date. This report describes, for the first time, the effective oral delivery of a second generation oligonucleotide where significant milligram amounts of intact drug are absorbed in human subjects. In this study, a variety of oral solid dosage formulations were evaluated and it was determined that pulsing the delivery of sodium caprate (C10), a well-known permeation enhancer, in a novel manner may provide optimal ASO plasma bioavailability. Further, these dosage forms, containing C10 and ASO, were well tolerated in both fasted and fed volunteers. Oral absorption of the 20 -O-(2-methoxyethyl) modified antisense oligonucleotide (20 -MOE ASO), ISIS 104838, was demonstrated in healthy volunteers with an average 9.5% plasma bioavailability across four formulations tested. The greatest average performance achieved in this study for a single formulation was 12.0% bioavailability within an individual dose and subject range of 1.96–27.5%. The totality of the data suggests that formulations can be devised that allow oral administration of oligonucleotides that maintain systemic concentrations associated with inhibition of targeted human mRNA. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:225–236, 2008
Keywords: vailability
antisense; oligonucleotides; pharmacokinetics; oral formulations; bioa-
INTRODUCTION Antisense oligonucleotides (ASOs) are synthetic oligonucleotides, usually between 15 and 25 bases in length that are designed to hybridize to mRNA through Watson-Crick base pairing. Upon binding to the target mRNA, the oligonucleotide inhibits expression of the encoded protein product in a sequence specific manner. With this specificity and the well-characterized rules for Watson-Crick base pairing, antisense technology offers great potential to be a simple approach for the rational design of effective therapeutics.1 Since the first report of antisense-mediated gene suppression in 1978,2 the interest in ASOs as pharmaceutical Correspondence to: Lloyd G. Tillman (Tel: 760 603 2586; Fax: 760 603 4655; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 225–236 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
agents for the treatment of cardiovascular disease, cancers, various infections, inflammatory and metabolic diseases has increased.1,3 While ASOs have attractive therapeutic potential their oral delivery is challenging in light of their physicochemical properties such as hydrophilicpolyanionic chemistry, high molecular weight and gastrointestinal (GI) instability. These factors have limited ASO administration to parenteral routes for systemic indications. The recent advent of novel oral delivery technologies described here, coupled with the added stability and tissue accumulation possible with the longer half-life provided by 20 -O-(2-methoxyethyl) partially modified oligonucleotides (20 -MOE ASO), allows orally administered ASOs to achieve and maintain therapeutic levels for selected systemic indications (Cook D., Monia B., Gapped Oligonucleotides. US Patent 7015315, Issued 2006). 20 MOE ASOs have an increased resistance to
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nuclease metabolism which enhances both gut stability4 and tissue accumulation.5 The difference in tissue accumulation between a typical phosphorothioate deoxy-ASO (tissue t½ 2 days) and 20 -MOE ASO (tissue t½ 15 days) equates to a seven- to eightfold higher tissue concentration for the 20 -MOE gapmer chemistry when dosed on an equivalent daily dosing regimen. Thus, with increased tissue accumulation, bioavailabilities of 10% or greater should be adequate to achieve systemic therapy. Oligonucleotides are highly water soluble therapeutics that have low membrane permeability and thus fall into Class III of the biopharmaceutic classification system.6,7 Like many other Class III compounds, they are not absorbed at all or only to a very small degree. This is not surprising as highly charged species resist partitioning across lipid bilayers and higher molecular weights are effectively restricted by the function of the tight junctions.8 In vitro and in vivo preclinical studies have examined the potential of using medium chain fatty acids with chain length of about 6–12 carbon atoms, as well as bile salts, to facilitate the absorption of poorly permeable compounds across the intestinal mucosa.9 The literature references sodium caprate (C10) being used as a permeation enhancer in both preclinical10 and clinical studies of Class III compounds.11 Permeation data from in situ rodent studies calculate tight junctions opening after C10 dosing which is consistent with the cross-sectional diameter of ASOs.12,13 The work of Raoof et al. demonstrated in the pig and dog, that the use of permeation enhancers, notably C10, represents an attractive strategy to enhance the oral delivery of ASO molecules.14,15 ISIS 104838 is a 20-base phosphorothioate (PS) oligonucleotide containing 20 -O-(2-methoxyethyl) modified nucleosides at each of the five terminal 30 and 50 nucleotide sugars. The structure of ISIS 104838 is shown in Figure 1. The central 10 phosphorothioate nucleotides located within the gap of MOE modified sugars are unmodified 20 -deoxyribose nucleotides allowing recruitment of RNaseH, a potent mechanism for suppression of mRNA translation. ISIS 104838 shows a sequence-specific suppression of TNF-a with an IC50 of <1 mM for inhibition of TNF-a mRNA in LPS-activated THP-1 monocytic cells.16 Excessive production of this cytokine has been implicated in the development and progression of many inflammatory, autoimmune and infectious diseases.17,18 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
The studies described herein are the first to report on the bioavailability and tolerability of solid dosage formulations of 20 -MOE modified ASOs administered orally to human volunteers.
MATERIALS AND METHODS Oral Formulations ISIS 104838 was manufactured by ISIS Pharmaceuticals, Inc. (Carlsbad, CA). To bracket the desired delivery rates of ISIS 104838 and C10, four different capsule formulations were manufactured by Division of Pharmaceutical Service, University of Iowa (Iowa City, IA). One capsule formulation contained a single population of 2 mm minitablets (Formulation A), while each of the other three capsule formulations had two populations of minitablets (Formulations B, C, and D) to allow for a second pulse of C10 to be delivered to the gut. The population of minitablets common to all formulations contained an immediate releasing (IR) presentation of oligonucleotide, C10 and, prior to irradiation, the stable isotope, samarium oxide which is further discussed below. The other minitablet population was either a short or long delayed release (DR) presentation of only C10. The DR minitablets were coated with different levels of a methacrylate polymer (Eudragit1 RS30D, Ro¨hm America, L.L.C., Piscataway, NJ) allowing for this short or long delayed pulse of the C10 component. Note that the delayed C10 pulse was designed to occur subsequent to the IR release of C10 from the uncoated, IR, population of minitablets. Formulation development efforts achieved this C10 pulse which effectively allowed for a lengthened ‘presentation’ of solubilized fatty acid in the gut. An in vitro view of this dissolution is shown in Figure 2 where a developmental batch of the longer delayed release capsule configuration is depicted by way of each of the two minitablet populations separately and also combined in a finished capsule. Natural abundance samarium oxide was included in the granule for the IR releasing minitablets to allow for in vivo temporal monitoring of capsule position and disintegration within the GI tract using gamma-scintigraphy. The gamma emitting radioisotope (153Sm) was produced by neutron irradiation of the already assembled and coated capsules 48 h prior to dosing. Manufacturing of the capsules was performed as follows: Minitablet DOI 10.1002/jps
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Figure 1. Chemical structure of ISIS 104838.
Figure 2. Dissolution profiles of C10 from two unencapsulated minitablet populations, immediate release (IR) and delayed release (DR), and from the finished enteric coated (EC) capsule containing both populations. Dissolution performed in 6.8 pH phosphate buffer at 378C with 50 rpm agitation by USP Method 1 paddles. Note that the IR minitabs contain both C10 and antisense oligo (ASO) while the DR minitabs only contain C10. DOI 10.1002/jps
portions were weighed and hand-filled into Size 000 hard gelatin capsules. Closed capsules were individually weighed prior to banding (Quali-Seal Laboratory Capsule Bander, Qualiseal, Indianapolis, IN). Banded capsules were enteric coated in a perforated pan coater (Accela Cota, Thomas Engineering, Hoffman Estates, IL) with a formulation containing Eudragit1 L30 D55. Prior to use, disintegration testing was carried out using 0.1 N HCl at 378C for 1 h, followed by pH 6.8 buffer in an EP disintegration apparatus with disks (QC21 Test System with EP basket, Hanson Research, Chatsworth, CA). All capsules were intact after 1 h disintegration in 0.1 N HCL. Neutron irradiation of the enteric coated capsules had no effect on capsule physical or chemical integrity as demonstrated by disintegration and analytical testing of capsules prior to and after the irradiation process (data not shown). A description of each of the formulations is given in Table 1. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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Table 1. Drug Products for Oral Administration of ISIS 104838
Formulation A B C D
Capsule Compositiona (ISIS 104838/C10)
ISIS 104838 (mg)
Description
100 100 100 140
Uncoated IR minitabs 50:50b IR:DR, 4% DR coating 50:50b IR:DR, 6% DR coating 70:30b IR:DR, 4% DR coating
100 100 100 140
mg/660 mg mg/660 mg mg /660 mg mg /660 mg
IR, immediate release; DR, delayed release. a All capsules were enteric coated. b Ratio denotes balance of C10 in uncoated IR minitabs and coated/delayed pulse minitabs.
Study Design and Procedures A five-way, open-label, partially randomized, crossover study was conducted with healthy male volunteers. The first (to accommodate scintigraphy) and fifth (to accommodate post-prandial dosing) administrations were non-randomized; the second, third and fourth administrations were randomized. Fifteen volunteers were dosed at the initiation of the study with the expectation that at least 12 would complete all treatment arms. Each treatment period was approximately 24 h in duration with a minimal washout period of 72 h between treatments. The allocation of treatments and volunteers are shown below in Table 2. The clinical portion of this study was conducted at Pharmaceutical Profiles Ltd. (Nottingham, UK) and approved by an independent ethics committee. The study was conducted in accordance with the ethical principles consistent with the Declaration of Helsinki and the International Conferences of Harmonization (ICH) Harmonized Tripartite Guidelines for Good Clinical Practice and local regulations relevant to the use of investigational therapeutic agents. At each treatment, subjects received five capsules containing minitablets of the respective formulations. Subjects were required to enter the testing facility the evening prior to drug administration and to remain in the testing facility for approximately 24 h post-drug administration. During this time, consumption of food containing caffeine or alcohol was prohibited and subjects consumed standard meals.
Subjects Healthy male volunteers (N ¼ 15) between 18 and 45 years with a body mass index of 18–30 kg/m2 were enrolled within 21 days before admission to the clinical site. After providing informed consent, demographic characteristics and medical/medicaJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
tion histories were recorded. Physical examination and vital signs were performed and blood/ urine samples were obtained for routine chemistry, hematology, coagulation testing (aPTT and PT) and urinalysis. Additional testing included a hepatitis B virus, hepatitis C virus and HIV test. In the routine screening laboratory test, negative serology results were required of all subjects. Standard breath alcohol and breath carbon monoxide tests were performed on all subjects. All Table 2. Treatment Allocation Study Visit Subject
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
A A A A A A A A A A A A A A A
C B C C C D D C C C D D D D B
B C D B D B B D B B B B C B D
D D B D B C C B D D C C B C C
E E E E E E E E E E E E E E E
(A) IR ISIS 104838/C10 minitabs in EC capsule (Formulation A) radiolabeled with 1 MBq 153Sm administered following an overnight fast. Following administration of this formulation, in vivo gamma scintigraphy was performed. (B) IR ISIS 104838/C10 minitabs plus DR (4% coating) C10 minitabs, at 50:50, administered following an overnight fast (Formulation B). (C) IR ISIS 104838/C10 minitabs plus DR (6% coating) C10 minitabs, at 50:50, administered following an overnight fast (Formulation C). (D) IR ISIS 104838/C10 minitabs plus DR (4% coating) C10 minitabs, at 70:30, administered following an overnight fast (Formulation D). (E) The best performing prototype of the first four regimens administered 2 h after receiving a high fat breakfast (Formulation D). DOI 10.1002/jps
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subjects had their urine tested for the presence of drugs of abuse before each dosing on each study day. If positive, and no therapeutic or other reasonable explanation was available, the subject was withdrawn from the study.
Dose Administration Each subject received five of the respective formulation capsules per treatment. Treatments A–D were administered in the morning with 240 mL of water, following an overnight fast. Each subject was required to drink an additional 180 mL of water at 2 h post-dose. Lunch was served 4 h post-dose and the evening meal about 9 h post-dose. Treatment E was administered 2 h after completion of a high fat breakfast [two fried eggs, two strips bacon, two slices of toast or bread/roll with butter, 4 oz hash brown potatoes, 150 mL whole milk] which was consumed within 30 min. Lunch was served about 6 h post-dose and the evening meal about 11 h post-dose.
Gamma Scintigraphic Procedure Anatomical markers containing 0.1 MBq 99mTc were taped to the skin anteriorly where the midclavicular line meets with the right costal margin so that these lay in approximately the same transverse plane as the pylorus. Anterior scintigraphic images, each 50 s duration were recorded using a gamma camera (General Electric Maxicamera) fitted with a low energy parallel hole collimator. Images of 50 s duration were recorded at approximately 10 min intervals until 12 h postdose and then every 20 min until 16 h post-dose. A final image was taken at 24 h post-dose. The subjects remained moderately active with freedom to walk around the clinical unit during the study period and all images were acquired with the subjects standing in front of the gamma camera. The images were recorded using a Park Medical computer system (Park Medical, Farnborough, UK) and were stored on digital audio tape for subsequent analysis. Since the dose of 153 Sm was localized to the minitablets, once the minitablets were released from the capsule and dispersed the signal strength was generally too low to detect. Thus, scintigraphic images provided information on capsule location only up to its disintegration within the gastrointestinal (GI) tract. DOI 10.1002/jps
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Blood Sampling and Bioanalysis Venous blood samples (5.9 mL) were withdrawn by cannula or direct venipuncture and collected into EDTA Sarstedt monovettes at each sampling time. For Treatments A–D samples were drawn pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, and 24 h post-dose. Following Treatment E, samples were drawn pre-dose and at 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 12, and 24 h post-dose. Following centrifugation at 3000 rpm, the resulting plasma was split into duplicate aliquots and stored frozen at 808C until analyzed. Samples were analyzed by PPD Development (Richmond, VA) using a hybridization ELISA method provided by ISIS Pharmaceuticals and previously validated at PPD Development. The lower limit of quantitation (LLOQ) for this assay was 0.77 ng/mL. The assay specificity has been validated for parent (20 mer) drug having zero percent cross-reactivity with the major metabolic products of ISIS 104838. This ELISA assay method is based on a modification of a previously published procedure.19,20 An interim analysis was performed on all plasma samples following the first four dosing days to determine the formulation to be used in the fed state (Treatment E). Safety Safety was assessed by monitoring for adverse events, physical examination, and results of routine blood chemistry, hematology, coagulation testing (aPTT and PT) and urinalysis. A follow-up assessment of all subjects was made within 14 days following the final dose. Data Analysis Scintigraphic Data. Gastric emptying time was determined as the mid-time between the times recorded for the two images about the transition of movement of the capsule from the stomach to the small intestine. Onset of initial capsule disintegration was defined as the time to detect any sign of release of the radioactive marker. Complete capsule disintegration was defined as the time at which all of the radioactive marker has dispersed in the GI tract and no signs of a distinct ‘‘core’’ remain. Anatomical location of initial capsule disintegration and anatomical location of complete capsule disintegration was also determined. Pharmacokinetic Data. All pharmacokinetic parameters were derived using WinNonlin Professional (Version 3.2, Pharsight Corporation, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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Mountain View, CA), using standard model independent methods. The pharmacokinetic parameters estimated were maximum plasma concentration (Cmax), time to reach Cmax (Tmax), lag time to beginning of absorption (Tlag, the time between administration and start of absorption), area under the plasma concentration–time curve (AUC) from zero time to the last measurable concentration (Clast) was calculated using the linear trapezoidal rule. Extrapolation to infinity (AUC1) was accomplished by adding Clast/lz to AUC0–Clast, where lz is the slope (decay rate constant) of the terminal plasma concentration–time curve. Relative plasma bioavailability (%F), was calculated by dividing the dose-normalized oral plasma AUC1 with the dose normalized historical parenteral plasma AUC1 for ISIS 104838 when dosed as a bolus s.c. injection at a dose of 25 mg16 using the following equation: Relative Plasma BAV ¼
AUCpo =Dosepo 100% AUCsc =Dosesc
Descriptive summary statistics were calculated for all subjects in each arm and for only those subjects for whom gastric emptying time occurred before 16 h post-administration as determined by gamma scintigraphy for Treatment A and by evidence of late absorbing drug (24 h) in Treatments B–E.
Statistical Analysis Descriptive statistics were calculated using standard methodology (Microsoft Excel). Differences in the pharmacokinetic parameters by formulation were tested using a nonparametric one-way ANOVA on ranks (Kruskal–Wallis) using SigmaStat (Version 2.0, SPSS, Inc., Chicago, IL).
study drug was well tolerated, with very few adverse events being reported. Headache and neck stiffness were the most common adverse events, both being reported by only two subjects. Review of laboratory data during the course of the study showed no abnormalities that were considered to be attributable to study medication. Figure 3 presents the gastric emptying time for each of the five capsules of ISIS 104838/C10 following administration of Treatment A. These data indicate that in the majority of subjects, under fasting conditions, the capsules were emptied from the stomach within 5 h. In four subjects (#8, 10, 12, and 15) a number of the capsules exhibited gastric emptying times greater than 16 h. Individual ISIS 104838 plasma concentration/time profiles for Treatment A are presented in Figure 4. When there was evidence of minitablets releasing after the predefined pharmacokinetic sampling scheme (gastric emptying time occurring >16 h post-administration), we observed Cmax values of <0.1 mg/mL and/or measurable drug concentrations at 24 h. On this basis pharmacokinetic data for these four subjects have been redacted from calculation of treatment averages. Gamma scintigraphy was not available for Treatment groups B through E. However, evidence of delayed gastric emptying can be inferred from pharmacokinetic profiles that exhibit measurable drug levels 24 h after oral administration, since no subject that exhibited early gastric emptying exhibited measurable plasma drug levels at 24 h. Mean plasma concentration–time profiles and descriptive statistical summary pharmacokinetic
RESULTS Fifteen subjects were enrolled into the study with different numbers completing each treatment. For Treatments A, N ¼ 15; for B through D, N ¼ 14; for Treatment E, N ¼ 12. None of the enrolled subjects had any significant findings in their medical histories or upon physical examination upon screening. One subject failed to return to the clinic following Treatment C (second visit: Subject No. 1), while two additional subjects (Subject Nos. 3 and 15) were lost to follow up in the interim period between Treatments D and E. Overall the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
Figure 3. Gastric emptying time for each of five capsules (single dose) by subject as determined by gamma scintigraphic tracking of the samarium radioisotope. Each diamond represents one capsule—Treatment A. DOI 10.1002/jps
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Figure 5. Mean ISIS 104838 concentration–time profiles in plasma following oral administration of various test formulations. Each data point represents the mean of 6–13 individual measurements and the error bars are standard deviation.
Figure 4. Spaghetti plots of ISIS 104836 plasma concentration–time profiles following administration of Treatment A.
parameters for ISIS 104838 by treatment are shown in Figure 5 and Table 3, respectively. Under fasted conditions mean absorption lag time (Tlag) and time to maximum plasma concentration (Tmax) were similar for all formulations. When administered following a high fat meal, a significant delay was observed in both these parameters compared to that under fasted conditions, although no significant differences were observed in the parameters Cmax or AUC. The plasma BAV relative to parenteral administration of ISIS 104838 by subject and treatment arm is presented in Table 4. Individual subject BAV of ISIS 104838 was highly variable and ranged from 0.7% to 27.5% across the entire data set when the formulations were administered following an overnight fast and exhibited complete stomach emptying prior to 16 h. Only 6 of 12 dosed subjects in Treatment E (post-fatty breakfast) exhibited evidence of complete stomach emptying in a manner that allowed for pharmaDOI 10.1002/jps
cokinetic assessment. For these six subjects, Cmax and AUC were similar to fasted state. Overall, two subjects were identified that exhibited a consistent pattern of delayed gastric emptying and subsequent capsule disintegration (Subjects #10 and 15) confirmed by both scintigraphy (Treatment A) and late (24 h) absorbing ASO (Treatments B–E). Assessment of the effect of gastric emptying time and anatomical site of disintegration on relative bioavailability in Treatment A is presented in graphic form (Figs. 6 and 7). There was no statistically significant effect for either parameter. However, there was a trend for decreased bioavailability with greater delay in gastric emptying as well as disintegration that occurred more distal to the stomach.
DISCUSSION Development of a successful formulation approach for delivery of ASOs orally is expected to be applicable across the technology platform. The applicability of the formulations across the platform derives from the fact that the pharmacokinetics of ASOs are similar across multiple ASO compounds independent of sequence differences and thus highly dependent upon ASO chemisJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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Table 3. Average Pharmacokinetic Parameters by Treatment Treatment Pharmacokinetic Parameter N (sample number) ISIS 104838 Dose (mg) Tlag (h) Tmax (h) Cmax (mg/mL) AUC1 (mg h/mL) %Bioavailabilityc
A
B
C
D
Ea
11 500 1.56 (0.22) 3.33 (0.20) 1.80 (0.35) 3.28 (0.60) 10.1 (1.96)
13 500 1.23 (0.27) 3.50 (0.27) 1.63 (0.22) 2.91 (0.40) 8.72 (1.20)
12 500 1.33 (0.14) 3.13 (0.31) 1.29 (0.29) 2.39 (0.64) 7.16 (1.93)
10 700 1.15 (0.15) 2.90 (0.46) 2.88 (0.61) 5.60 (1.30) 12.0 (2.78)
6 700 4.25 (0.54)b 6.75 (1.33)b 2.27 (0.64) 6.01 (1.42) 12.9 (3.93)
N, number of subjects included in average. Each average is comprised of 6–13 individuals. Each pharmacokinetic parameter estimate is presented as the average (standard error). a Post-prandial dosing, 2 h following a fatty breakfast. b Significantly delayed ( p < 0.05). c Relative bioavailability as compared to a subcutaneous dose of 25 mg ISIS 104838.
try.21 The development of the second generation chemistries, notably the 20 -O-(2-methoxyethyl) modification in the nucleosides at each of the five terminal 30 and 50 nucleotide sugars of PS oligonucleotides has led to compounds which have an increased binding affinity for the target mRNA and resist endogenous exonucleases thereby prolonging drug half-life.5,16 ISIS 104838 is an example of this new class of modified phosphor-
othioate ASOs involving partial 20 -alkoxy modifications of the ribose sugars in the backbone of the oligonucleotide. These initial results in human volunteers provide guidance for further oral development applicable to other ASOs in this chemical class. The prototype oral formulations presented in this study included two primary components: (1) enteric coating of the filled capsule and (2) a
Table 4. Relative Plasma Bioavailability (%) Summary by Subject (Male Volunteers) and Treatment (Formulation) Treatment Subject# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Average standard deviation (all) Average standard deviationb
A
B
C
D
Ea
13.9 3.51 13.0 8.83 1.72 4.90 21.5 1.89 16.2 0.02 1.70 7.13 11.5 14.0 1.82 8.11 6.59 10.1 6.49
Not dosed 4.12 3.97 15.1 5.85 5.42 14.0 9.03 2.16 12.7 6.95 10.8 14.1 9.35 1.29 8.19 4.62 8.72 4.34
6.28 0.67 4.23 10.8 7.53 1.06 25.9 6.75 0.22 0.73 2.99 6.08 3.69 9.93 Not dosed 6.21 6.62 7.16 6.69
Not dosed 1.96 5.39 3.88 13.9 1.89 18.8 27.5 2.01 1.20 3.11 18.8 19.1 7.63 1.45 9.04 8.78 12.0 8.78
Not dosed 0.03 Not dosed 0.08 1.73 12.40 28.1 0.65 1.30 1.28 4.73 15.6 15.8 3.01 Not dosed 7.05 8.92 12.9 9.62
Treatment A, novel rapid IR minitablets; Treatment B, pulsatile minitablets (50/50 rapid pulse); Treatment C, pulsatile minitablets (50/50 slow pulse); Treatment D, pulsatile minitablets (70/30 rapid pulse); Treatment E, pulsatile minitablets (70/30 rapid pulse) dosed 2 h after fatty breakfast. a Fatty breakfast fed arm (2 h post-prandial dosing), all other arms were fasted overnight. b Redacted average ¼ removed AUCs () for which gastric emptying of enteric capsules occurred after 16 h post-administration) as determined by gamma scintigraphy for Treatment A and by evidence of late absorbing drug (24 h) in Treatments B–E. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
DOI 10.1002/jps
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Figure 6. Bioavailability appears to decrease as the ‘‘delta GE’’ (measured by determining the difference in time between first and last capsule leaving the stomach) increases beyond 1 h. Analysis is not powered to provide statistical significance. Each symbol represents one subject. Subjects 8, 10, 12, and 15 were not included in this analysis (some capsules did not leave stomach during the scintigraphic measurement period) since plasma profiles are not available for the late emptying capsules.
series of ‘pulsatile release’ dosage forms designed to give immediate release of the oligonucleotide concurrent with a measured amount of C10 (true for all formulations) followed by a second pulsed
Figure 7. Anatomical position for Treatment A capsule disintegration as determined by gamma scintigraphy and its effect on plasma bioavailability of ISIS 104838. Each point represents one subject. Subjects 8, 10, 12, and 15 were not included in the analysis since scintigraphy data was not available for late gastric emptying capsules. DOI 10.1002/jps
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release of the balance of C10 component (Formulations B, C, and D). The requirement of the enteric coat relates to the properties of both the oligonucleotide and the permeation enhancing excipient, C10, in that ISIS 104838 and other oligonucleotides of similar chemistry are susceptible to acid catalyzed depurination while C10 is converted to its unionized acid form which is an insoluble oil at 378C. The pulsatile release component incorporated in these prototype formulations—immediate release (IR) followed by a delayed release (DR)—maximizes oligonucleotide uptake by prolonging the luminal presence of the C10 component at concentrations relating to permeation action. In effect, the two pulses of C10 sustain, for a brief duration, the C10 luminal concentrations as it is rapidly cleared from the small intestine by both dilution and absorption mechanisms. Putatively, this longer C10 residence time within the gut lumen allows for a prolonged duration of membrane permeability and extended mucosal surface area for the oligonucleotide to be absorbed. This formulation approach is slightly different from the synchronous release of C10 and drug that has been proposed in the literature by Baluom et al.22,23 In contrast, the formulations tested in this study are designed to: initially establish high concentrations of both oligonucleotide and C10 followed by a continued or extended release of supplemental C10. This design takes advantage of the firstorder absorption properties of oligonucleotides which are optimized with high concentrations of oligonucleotide in the presence of active concentrations of C10. Put another way, the oligonucleotide absorption process is far more effective if all of the oligonucleotide is in solution and at high luminal concentration during the brief window available for absorption—since the rate of absorption would be dependent on the oligonucleotide concentration (data not shown). As described above, all four capsule formulations tested in this study had an immediate releasing minitablet population resulting in a fast release of the oligonucleotide and C10. One of these formulations (Formulation A) only had this IR minitablet population and therefore served as a ‘‘single pulse’’ control for the remaining formulations. Among the three formulations having delayed releasing (DR) C10 minitablets, the second pulse of C10 was designed to have a delay of either 20 or 40 min with the 20 min delay termed as a ‘‘rapid pulsatile’’ and the 40 min delay termed ‘‘slow pulsatile’’. It’s important to note JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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here that after minitablet encapsulation and enteric coating of the intact capsule it is clear that an additional lag time is evident as seen in Figure 2 where the 40 min C10 release was not complete until approximately 60 min. The results of the study suggest that the ‘‘rapid’’ C10 pulse release may provide some improvement in overall systemic bioavailability (Formulation D; used in Treatments D and E) wherein 70% of the C10 and 100% of the oligonucleotide is released in the IR portion followed by the remaining 30% of the C10. While the trend for improvement compared to the single IR formulation (A) and the two other two-pulse formulations (B and C) does exist, the increase is small and not statistically significant. Pharmacokinetic studies of the 20 -O-(2-methoxyethyl) modified PS oligonucleotides in both animals and man following intravenous and subcutaneous administration have led to the following observations: (1) the plasma pharmacokinetics of ISIS 104838 following parenteral administration is descriptive of distribution; plasma clearance being dominated by tissue distribution,5 (2) the plasma pharmacokinetics are somewhat nonlinear with increasing doses exhibiting somewhat greater than dose proportional increases in AUC and with more rapid distribution at doses of 0.5 mg/kg or less,16 (3) they are slowly cleared from tissue by relatively slow nuclease metabolism and efflux into plasma followed by urinary excretion of the shortened metabolite products, and (4) S.C. administration results in essentially complete absorption of oligonucleotide.5 The known nonlinear distribution component of oligonucleotide pharmacokinetics was factored into the selection of an appropriate reference parenteral dose. The historical (external control) parenteral dose selected (25 mg) represents 5% of the delivered oral dose and produces similar plasma concentrations to those observed following oral administration at a dose of 500 mg. Parenteral pharmacokinetics of oligonucleotides in general and specifically for ISIS 104838 is well behaved with reproducibility across studies and without observed differences between normal volunteers and patients in numerous clinical studies (unpublished comparisons, ISIS Pharmaceuticals, Inc.). Thus, with the primary goal of this study being the comparison of numerous formulation prototypes, an external control at an exposure-equivalent dose administered subcutaneously (s.c.) in accordance with current clinical practice for ISIS 104838, and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
other ASOs in this chemical class, was deemed appropriate. The use of gamma-scintigraphy allowed visualization of the movement of the minitablets throughout the GI tract of the male volunteers and permitted evaluation of gastric emptying and gastrointestinal transit on the performance of the IR ASO formulation. Using these data we were able to correlate the observed delayed gastric emptying of the minitablets in a subset of subjects with late absorbing drug, which allowed for the identification of delayed gastric emptying subjects based on pharmacokinetics alone for the remaining prototypes that were tested without scintigraphy. The reduced apparent bioavailability observed for late gastric emptying of capsules is likely an artifact of the pharmacokinetic sampling schedule such that any oligonucleotide released from the capsules and absorbed after sampling is complete would not be included in the measured AUC. In addition to identification of late gastric emptying subjects, the gammascintigraphy data also provide confidence that oligonucleotide was absorbed from all regions of the small intestine without significant limitations. While there was an apparent trend for lower absorption in the distal small intestine compared with proximal small intestine, the number of observations does not provide appropriate power and conclusions in this regard should be taken with caution. Plasma concentration data taken after oral administration tends to underestimate the endorgan bioavailability of ISIS 104838.14 In a study in dogs, Raoof et al.14 determined the tolerability and pharmacokinetics of ISIS 104838 following multi-dose administration by intravenous and oral routes. In this dog study, an enteric-coated tablet formulation of ISIS 104838 (80 mg) and C10 (330 mg) was administered by oral gavage daily for five days. The average plasma BAV, based on the ratio of plasma AUC Oral: IV averaged 1.4%. The tissue distribution profile was similar following either route of administration, and the mean systemic target tissue BAV ranging from 2.0% to 4.3%, relative to tissues following IV administration. Distribution was dependent on the tissue type with the highest concentrations of intact oligonucleotide achieved in the kidney and liver. These organs demonstrated the highest and most consistent bioavailability as well. For antisense drugs, such as ISIS 104838, the tissue bioavailability determines pharmacological activity 24 since the target is intracellular mRNA. DOI 10.1002/jps
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Based on the nonclinical studies discussed above, it seems reasonable to posit that the average relative plasma bioavailability of 7–13% attained in this study with the various formulations represents an underestimate of the actual tissue bioavailability of ISIS 104838. While the effective plasma distribution half-life of 20 -O-(2methoxyethyl) modified PS oligonucleotides is approximately 1–2 h, for ISIS 104838 a prolonged phase with a terminal half-life of about 27 days has been identified16 that likely reflects the return of ISIS 104838 from tissue stores to plasma where it is then rapidly eliminated. This suggests that tissue accumulation upon repeat daily administration will be substantial. These two properties, the underestimation of true tissue bioavailability from plasma data and the prolonged tissue halflife of ISIS 104838, provide good confidence that these prototype formulations will deliver active amounts of ASO to the target tissues. The long half-life also mitigates variability in bioavailability. The high degree of variability observed in this study is substantial. We hypothesize that the observed variability in bioavailability within and between subjects will be reflected in routine multiple dose administration. When modeled using first-order pharmacokinetic models, integrating a random bioavailability from day to day, the accumulation in tissue at steady-state reflected the average bioavailability over a period of several months. In other words, variability in daily bioavailability was mitigated by the long half-life yielding ultimate tissue levels that are predicted to be relatively stable at steady-state. In conclusion, the combination of an absorption enhancer (sodium caprate), the 20 -O-(2-methoxyethyl) modified PS oligonucleotide (ISIS 104838) in a multiparticulate, minitablet presentation, and delivered within enteric coated gelatin capsules yielded measurable plasma concentrations for ISIS 104838 in all healthy male volunteer subjects. Based on the current study, oral dosing of ISIS 104838 results in measurable plasma bioavailability of at least 10% relative to subcutaneous administration. It is likely that the plasma bioavailability underestimates the true tissue bioavailability of this compound based on preclinical evaluations. These data taken together with our previous preclinical experience provide confidence that clinical oral application would be successful for modified ASOs. The value of this technology is clear when one considers that positive pharmacology of this class of oligonucleotide is demonstrated with dosing regimens as low DOI 10.1002/jps
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or lower than 200 mg administered weekly by the subcutaneous route.25 With oral dosing of about 400 mg daily—from four capsules as formulated in this study where approximately 10% bioavailability would be expected—the systemic exposure should exceed that required for therapeutic efficacy.
ACKNOWLEDGMENTS This work was supported by Orasense Ltd., a joint venture between ISIS Pharmaceuticals, Inc. and Elan Pharmaceuticals. As of JUN2004, Orasense is now a wholly owned subsidiary of ISIS Pharmaceuticals, Inc. We thank Dr. Tony Fitzpatrick and Triona Healy of Elan Pharmaceuticals for management and oversight of this trial and Dr. A.L. Connor and Dr. Andrew Salmon, for their assistance in the scientific and medical conduct of the study at PPL. We thank Dr. Rosie Yu, Dr. Arthur Levin and Dr. Stanley Crooke for their expert review and helpful discussions. Special thanks to Robert Saunders for his expert assistance in the preparation of this manuscript.
REFERENCES 1. Bennett CF, Butler M, Cook PD, Geary RS, Levin AA, Mehta R, Teng C-L, Deshmukh H, Tillman L, Hardee G. 2000. Antisense oligonucleotide-based therapeutics. In: Templeton NS, Lasic DD, editors. Gene therapy, first edition. New York: Marcel Dekker, Inc. pp 305–332. 2. Zamecnik PC, Stephenson ML. 1978. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA 75:280–284. 3. Crooke ST, editor. 2001. Antisense drug technology: Basic principles, strategies, and applications, first edition. New York: Marcel Dekker, Inc. 4. Geary RS, Khatsenko O, Bunker K, Crooke R, Moore M, Burckin T, Truong L, Sasmor H, Levin AA. 2001. Absolute bioavailability of 2(-O-(2-methoxyethyl)-modified antisense oligonucleotides following intraduodenal instillation in rats. J Pharmacol Exp Ther 296:898–904. 5. Geary RS, Yu RZ, Watanabe T, Henry SP, Hardee GE, Chappell A, Matson J, Sasmor H, Cummins L, Levin AA. 2003. Pharmacokinetics of a tumor necrosis factor-alpha phosphorothioate 2(-O-(2-methoxyethyl) modified antisense oligonucleotide: Comparison across species. Drug Metab Dispos 31:1419–1428. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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6. FDA. 2000. Guidance for Industry on Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System. 7. Amidon GL, Lennernas H, Shah VP, Crison JR. 1995. A theoretical basis for a biopharmaceutic drug classification: The correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm Res 12:413–420. 8. Lindmark T, Kimura Y, Artursson P. 1998. Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells. J Pharmacol Exp Ther 284:362–369. 9. Aungst BJ. 2000. Intestinal permeation enhancers. J Pharm Sci 89:429–442. 10. Ishizawa T, Hayashi M, Awazu S. 1987. Enhancement of jejunal and colonic absorption of fosfomycin by promoters in the rat. J Pharm Pharmacol 39: 892–895. 11. Lindmark T, Soderholm JD, Olaison G, Alvan G, Ocklind G, Artursson P. 1997. Mechanism of absorption enhancement in humans after rectal administration of ampicillin in suppositories containing sodium caprate. Pharm Res 14:930–935. 12. Tsutsumi K, Li SK, Ghanem AH, Ho NF, Higuchi WI. 2003. A systematic examination of the in vitro using chamber and the in situ single-pass perfusion model systems in rat ileum permeation of model solutes. J Pharm Sci 92:344–359. 13. Ma TY, Hollander D, Bhalla D, Nguyen H, Krugliak P. 1992. IEC-18, a nontransformed small intestinal cell line for studying epithelial permeability. J Lab Clin Med 120:329–341. 14. Raoof AA, Chiu P, Ramtoola Z, Cumming IK, Teng C, Weinbach SP, Hardee GE, Levin AA, Geary RS. 2004. Oral bioavailability and multiple dose tolerability of an antisense oligonucleotide tablet formulated with sodium caprate. J Pharm Sci 93: 1431–1439. 15. Raoof AA, Ramtoola Z, McKenna B, Yu RZ, Hardee G, Geary RS. 2002. Effect of sodium caprate on the intestinal absorption of two modified antisense oligonucleotides in pigs. Eur J Pharm Sci 17:131–138. 16. Sewell LK, Geary RS, Baker BF, Glover JM, Mant TGK, Yu RZ, Tami JA, Dorr AF. 2002. Phase I trial of ISIS 104838, a 2(-methoxyethyl modified anti-
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sense oligonucleotide targeting tumor necrosis factor-alpha. J Pharmacol Exp Ther 303:1334– 1343. Aggarwal BB, Natarajan K. 1996. Tumor necrosis factors: Developments during the last decade. Eur Cytokine Netw 7:93–124. Beutler BA. 1999. The role of tumor necrosis factor in health and disease. J Rheumatol Suppl 57:16–21. Yu RZ, Geary RS, Levin AA. 2004. Application of novel quantitative bioanalytical methods for pharmacokinetic and pharmacokinetic/pharmacodynamic assessments of antisense oligonucleotides. Curr Opin Drug Discov Dev 7:195–203. Yu RZ, Baer B, Chappel A, Geary RS, Chueng E, Levin AA. 2002. Development of an ultrasensitive noncompetitive hybridization-ligation enzyme-linked immunosorbent assay for the determination of phosphorothioate oligodeoxynucleotide in plasma. Anal Biochem 304:19–25. Geary RS, Leeds JM, Henry SP, Monteith DK, Levin AA. 1997. Antisense oligonucleotide inhibitors for the treatment of cancer. 1. Pharmacokinetic properties of phosphorothioate oligodeoxynucleotides. Anticancer Drug Des 12:383–393. Baluom M, Friedman M, Rubinstein A. 1998. The importance of intestinal residence time of absorption enhancer on drug absorption and implication on formulative considerations. Int J Pharm 176: 21–30. Baluom M, Friedman M, Assaf P, Haj-Yehia AI, Rubinstein A. 2000. Synchronized release of sulpiride and sodium decanoate from HPMC matrices: A rational approach to enhance sulpiride absorption in the rat intestine. Pharm Res 17:1071–1076. Yu RZ, Zhang H, Geary RS, Graham M, Masarjian L, Lemonidis K, Crooke R, Dean NM, Levin AA. 2001. Pharmacokinetics and pharmacodynamics of an antisense phosphorothioate oligonucleotide targeting Fas mRNA in mice. J Pharmacol Exp Ther 296:388–395. Kastelein JJP, Wedel MK, Baker BF, Su J, Bradley JD, Yu RZ, Chuang E, Graham MJ, Crooke RM. 2006. Potent reduction of apolipoprotein B and low-density lipoprotein cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B. Circulation 114:1729–1735.
DOI 10.1002/jps
Received 18 October 2006; revised 20 December 2006; accepted 17 January 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21084
ABSTRACT: Treatment of systemic disease with phosphorothioate antisense oligonucleotides (PS ASOs) has been accomplished using local or parenteral routes of administration to date. This report describes, for the first time, the effective oral delivery of a second generation oligonucleotide where significant milligram amounts of intact drug are absorbed in human subjects. In this study, a variety of oral solid dosage formulations were evaluated and it was determined that pulsing the delivery of sodium caprate (C10), a well-known permeation enhancer, in a novel manner may provide optimal ASO plasma bioavailability. Further, these dosage forms, containing C10 and ASO, were well tolerated in both fasted and fed volunteers. Oral absorption of the 20 -O-(2-methoxyethyl) modified antisense oligonucleotide (20 -MOE ASO), ISIS 104838, was demonstrated in healthy volunteers with an average 9.5% plasma bioavailability across four formulations tested. The greatest average performance achieved in this study for a single formulation was 12.0% bioavailability within an individual dose and subject range of 1.96–27.5%. The totality of the data suggests that formulations can be devised that allow oral administration of oligonucleotides that maintain systemic concentrations associated with inhibition of targeted human mRNA. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:225–236, 2008
Keywords: vailability
antisense; oligonucleotides; pharmacokinetics; oral formulations; bioa-
INTRODUCTION Antisense oligonucleotides (ASOs) are synthetic oligonucleotides, usually between 15 and 25 bases in length that are designed to hybridize to mRNA through Watson-Crick base pairing. Upon binding to the target mRNA, the oligonucleotide inhibits expression of the encoded protein product in a sequence specific manner. With this specificity and the well-characterized rules for Watson-Crick base pairing, antisense technology offers great potential to be a simple approach for the rational design of effective therapeutics.1 Since the first report of antisense-mediated gene suppression in 1978,2 the interest in ASOs as pharmaceutical Correspondence to: Lloyd G. Tillman (Tel: 760 603 2586; Fax: 760 603 4655; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 225–236 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
agents for the treatment of cardiovascular disease, cancers, various infections, inflammatory and metabolic diseases has increased.1,3 While ASOs have attractive therapeutic potential their oral delivery is challenging in light of their physicochemical properties such as hydrophilicpolyanionic chemistry, high molecular weight and gastrointestinal (GI) instability. These factors have limited ASO administration to parenteral routes for systemic indications. The recent advent of novel oral delivery technologies described here, coupled with the added stability and tissue accumulation possible with the longer half-life provided by 20 -O-(2-methoxyethyl) partially modified oligonucleotides (20 -MOE ASO), allows orally administered ASOs to achieve and maintain therapeutic levels for selected systemic indications (Cook D., Monia B., Gapped Oligonucleotides. US Patent 7015315, Issued 2006). 20 MOE ASOs have an increased resistance to
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nuclease metabolism which enhances both gut stability4 and tissue accumulation.5 The difference in tissue accumulation between a typical phosphorothioate deoxy-ASO (tissue t½ 2 days) and 20 -MOE ASO (tissue t½ 15 days) equates to a seven- to eightfold higher tissue concentration for the 20 -MOE gapmer chemistry when dosed on an equivalent daily dosing regimen. Thus, with increased tissue accumulation, bioavailabilities of 10% or greater should be adequate to achieve systemic therapy. Oligonucleotides are highly water soluble therapeutics that have low membrane permeability and thus fall into Class III of the biopharmaceutic classification system.6,7 Like many other Class III compounds, they are not absorbed at all or only to a very small degree. This is not surprising as highly charged species resist partitioning across lipid bilayers and higher molecular weights are effectively restricted by the function of the tight junctions.8 In vitro and in vivo preclinical studies have examined the potential of using medium chain fatty acids with chain length of about 6–12 carbon atoms, as well as bile salts, to facilitate the absorption of poorly permeable compounds across the intestinal mucosa.9 The literature references sodium caprate (C10) being used as a permeation enhancer in both preclinical10 and clinical studies of Class III compounds.11 Permeation data from in situ rodent studies calculate tight junctions opening after C10 dosing which is consistent with the cross-sectional diameter of ASOs.12,13 The work of Raoof et al. demonstrated in the pig and dog, that the use of permeation enhancers, notably C10, represents an attractive strategy to enhance the oral delivery of ASO molecules.14,15 ISIS 104838 is a 20-base phosphorothioate (PS) oligonucleotide containing 20 -O-(2-methoxyethyl) modified nucleosides at each of the five terminal 30 and 50 nucleotide sugars. The structure of ISIS 104838 is shown in Figure 1. The central 10 phosphorothioate nucleotides located within the gap of MOE modified sugars are unmodified 20 -deoxyribose nucleotides allowing recruitment of RNaseH, a potent mechanism for suppression of mRNA translation. ISIS 104838 shows a sequence-specific suppression of TNF-a with an IC50 of <1 mM for inhibition of TNF-a mRNA in LPS-activated THP-1 monocytic cells.16 Excessive production of this cytokine has been implicated in the development and progression of many inflammatory, autoimmune and infectious diseases.17,18 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
The studies described herein are the first to report on the bioavailability and tolerability of solid dosage formulations of 20 -MOE modified ASOs administered orally to human volunteers.
MATERIALS AND METHODS Oral Formulations ISIS 104838 was manufactured by ISIS Pharmaceuticals, Inc. (Carlsbad, CA). To bracket the desired delivery rates of ISIS 104838 and C10, four different capsule formulations were manufactured by Division of Pharmaceutical Service, University of Iowa (Iowa City, IA). One capsule formulation contained a single population of 2 mm minitablets (Formulation A), while each of the other three capsule formulations had two populations of minitablets (Formulations B, C, and D) to allow for a second pulse of C10 to be delivered to the gut. The population of minitablets common to all formulations contained an immediate releasing (IR) presentation of oligonucleotide, C10 and, prior to irradiation, the stable isotope, samarium oxide which is further discussed below. The other minitablet population was either a short or long delayed release (DR) presentation of only C10. The DR minitablets were coated with different levels of a methacrylate polymer (Eudragit1 RS30D, Ro¨hm America, L.L.C., Piscataway, NJ) allowing for this short or long delayed pulse of the C10 component. Note that the delayed C10 pulse was designed to occur subsequent to the IR release of C10 from the uncoated, IR, population of minitablets. Formulation development efforts achieved this C10 pulse which effectively allowed for a lengthened ‘presentation’ of solubilized fatty acid in the gut. An in vitro view of this dissolution is shown in Figure 2 where a developmental batch of the longer delayed release capsule configuration is depicted by way of each of the two minitablet populations separately and also combined in a finished capsule. Natural abundance samarium oxide was included in the granule for the IR releasing minitablets to allow for in vivo temporal monitoring of capsule position and disintegration within the GI tract using gamma-scintigraphy. The gamma emitting radioisotope (153Sm) was produced by neutron irradiation of the already assembled and coated capsules 48 h prior to dosing. Manufacturing of the capsules was performed as follows: Minitablet DOI 10.1002/jps
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Figure 1. Chemical structure of ISIS 104838.
Figure 2. Dissolution profiles of C10 from two unencapsulated minitablet populations, immediate release (IR) and delayed release (DR), and from the finished enteric coated (EC) capsule containing both populations. Dissolution performed in 6.8 pH phosphate buffer at 378C with 50 rpm agitation by USP Method 1 paddles. Note that the IR minitabs contain both C10 and antisense oligo (ASO) while the DR minitabs only contain C10. DOI 10.1002/jps
portions were weighed and hand-filled into Size 000 hard gelatin capsules. Closed capsules were individually weighed prior to banding (Quali-Seal Laboratory Capsule Bander, Qualiseal, Indianapolis, IN). Banded capsules were enteric coated in a perforated pan coater (Accela Cota, Thomas Engineering, Hoffman Estates, IL) with a formulation containing Eudragit1 L30 D55. Prior to use, disintegration testing was carried out using 0.1 N HCl at 378C for 1 h, followed by pH 6.8 buffer in an EP disintegration apparatus with disks (QC21 Test System with EP basket, Hanson Research, Chatsworth, CA). All capsules were intact after 1 h disintegration in 0.1 N HCL. Neutron irradiation of the enteric coated capsules had no effect on capsule physical or chemical integrity as demonstrated by disintegration and analytical testing of capsules prior to and after the irradiation process (data not shown). A description of each of the formulations is given in Table 1. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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Table 1. Drug Products for Oral Administration of ISIS 104838
Formulation A B C D
Capsule Compositiona (ISIS 104838/C10)
ISIS 104838 (mg)
Description
100 100 100 140
Uncoated IR minitabs 50:50b IR:DR, 4% DR coating 50:50b IR:DR, 6% DR coating 70:30b IR:DR, 4% DR coating
100 100 100 140
mg/660 mg mg/660 mg mg /660 mg mg /660 mg
IR, immediate release; DR, delayed release. a All capsules were enteric coated. b Ratio denotes balance of C10 in uncoated IR minitabs and coated/delayed pulse minitabs.
Study Design and Procedures A five-way, open-label, partially randomized, crossover study was conducted with healthy male volunteers. The first (to accommodate scintigraphy) and fifth (to accommodate post-prandial dosing) administrations were non-randomized; the second, third and fourth administrations were randomized. Fifteen volunteers were dosed at the initiation of the study with the expectation that at least 12 would complete all treatment arms. Each treatment period was approximately 24 h in duration with a minimal washout period of 72 h between treatments. The allocation of treatments and volunteers are shown below in Table 2. The clinical portion of this study was conducted at Pharmaceutical Profiles Ltd. (Nottingham, UK) and approved by an independent ethics committee. The study was conducted in accordance with the ethical principles consistent with the Declaration of Helsinki and the International Conferences of Harmonization (ICH) Harmonized Tripartite Guidelines for Good Clinical Practice and local regulations relevant to the use of investigational therapeutic agents. At each treatment, subjects received five capsules containing minitablets of the respective formulations. Subjects were required to enter the testing facility the evening prior to drug administration and to remain in the testing facility for approximately 24 h post-drug administration. During this time, consumption of food containing caffeine or alcohol was prohibited and subjects consumed standard meals.
Subjects Healthy male volunteers (N ¼ 15) between 18 and 45 years with a body mass index of 18–30 kg/m2 were enrolled within 21 days before admission to the clinical site. After providing informed consent, demographic characteristics and medical/medicaJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
tion histories were recorded. Physical examination and vital signs were performed and blood/ urine samples were obtained for routine chemistry, hematology, coagulation testing (aPTT and PT) and urinalysis. Additional testing included a hepatitis B virus, hepatitis C virus and HIV test. In the routine screening laboratory test, negative serology results were required of all subjects. Standard breath alcohol and breath carbon monoxide tests were performed on all subjects. All Table 2. Treatment Allocation Study Visit Subject
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
A A A A A A A A A A A A A A A
C B C C C D D C C C D D D D B
B C D B D B B D B B B B C B D
D D B D B C C B D D C C B C C
E E E E E E E E E E E E E E E
(A) IR ISIS 104838/C10 minitabs in EC capsule (Formulation A) radiolabeled with 1 MBq 153Sm administered following an overnight fast. Following administration of this formulation, in vivo gamma scintigraphy was performed. (B) IR ISIS 104838/C10 minitabs plus DR (4% coating) C10 minitabs, at 50:50, administered following an overnight fast (Formulation B). (C) IR ISIS 104838/C10 minitabs plus DR (6% coating) C10 minitabs, at 50:50, administered following an overnight fast (Formulation C). (D) IR ISIS 104838/C10 minitabs plus DR (4% coating) C10 minitabs, at 70:30, administered following an overnight fast (Formulation D). (E) The best performing prototype of the first four regimens administered 2 h after receiving a high fat breakfast (Formulation D). DOI 10.1002/jps
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subjects had their urine tested for the presence of drugs of abuse before each dosing on each study day. If positive, and no therapeutic or other reasonable explanation was available, the subject was withdrawn from the study.
Dose Administration Each subject received five of the respective formulation capsules per treatment. Treatments A–D were administered in the morning with 240 mL of water, following an overnight fast. Each subject was required to drink an additional 180 mL of water at 2 h post-dose. Lunch was served 4 h post-dose and the evening meal about 9 h post-dose. Treatment E was administered 2 h after completion of a high fat breakfast [two fried eggs, two strips bacon, two slices of toast or bread/roll with butter, 4 oz hash brown potatoes, 150 mL whole milk] which was consumed within 30 min. Lunch was served about 6 h post-dose and the evening meal about 11 h post-dose.
Gamma Scintigraphic Procedure Anatomical markers containing 0.1 MBq 99mTc were taped to the skin anteriorly where the midclavicular line meets with the right costal margin so that these lay in approximately the same transverse plane as the pylorus. Anterior scintigraphic images, each 50 s duration were recorded using a gamma camera (General Electric Maxicamera) fitted with a low energy parallel hole collimator. Images of 50 s duration were recorded at approximately 10 min intervals until 12 h postdose and then every 20 min until 16 h post-dose. A final image was taken at 24 h post-dose. The subjects remained moderately active with freedom to walk around the clinical unit during the study period and all images were acquired with the subjects standing in front of the gamma camera. The images were recorded using a Park Medical computer system (Park Medical, Farnborough, UK) and were stored on digital audio tape for subsequent analysis. Since the dose of 153 Sm was localized to the minitablets, once the minitablets were released from the capsule and dispersed the signal strength was generally too low to detect. Thus, scintigraphic images provided information on capsule location only up to its disintegration within the gastrointestinal (GI) tract. DOI 10.1002/jps
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Blood Sampling and Bioanalysis Venous blood samples (5.9 mL) were withdrawn by cannula or direct venipuncture and collected into EDTA Sarstedt monovettes at each sampling time. For Treatments A–D samples were drawn pre-dose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, and 24 h post-dose. Following Treatment E, samples were drawn pre-dose and at 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7, 8, 12, and 24 h post-dose. Following centrifugation at 3000 rpm, the resulting plasma was split into duplicate aliquots and stored frozen at 808C until analyzed. Samples were analyzed by PPD Development (Richmond, VA) using a hybridization ELISA method provided by ISIS Pharmaceuticals and previously validated at PPD Development. The lower limit of quantitation (LLOQ) for this assay was 0.77 ng/mL. The assay specificity has been validated for parent (20 mer) drug having zero percent cross-reactivity with the major metabolic products of ISIS 104838. This ELISA assay method is based on a modification of a previously published procedure.19,20 An interim analysis was performed on all plasma samples following the first four dosing days to determine the formulation to be used in the fed state (Treatment E). Safety Safety was assessed by monitoring for adverse events, physical examination, and results of routine blood chemistry, hematology, coagulation testing (aPTT and PT) and urinalysis. A follow-up assessment of all subjects was made within 14 days following the final dose. Data Analysis Scintigraphic Data. Gastric emptying time was determined as the mid-time between the times recorded for the two images about the transition of movement of the capsule from the stomach to the small intestine. Onset of initial capsule disintegration was defined as the time to detect any sign of release of the radioactive marker. Complete capsule disintegration was defined as the time at which all of the radioactive marker has dispersed in the GI tract and no signs of a distinct ‘‘core’’ remain. Anatomical location of initial capsule disintegration and anatomical location of complete capsule disintegration was also determined. Pharmacokinetic Data. All pharmacokinetic parameters were derived using WinNonlin Professional (Version 3.2, Pharsight Corporation, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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Mountain View, CA), using standard model independent methods. The pharmacokinetic parameters estimated were maximum plasma concentration (Cmax), time to reach Cmax (Tmax), lag time to beginning of absorption (Tlag, the time between administration and start of absorption), area under the plasma concentration–time curve (AUC) from zero time to the last measurable concentration (Clast) was calculated using the linear trapezoidal rule. Extrapolation to infinity (AUC1) was accomplished by adding Clast/lz to AUC0–Clast, where lz is the slope (decay rate constant) of the terminal plasma concentration–time curve. Relative plasma bioavailability (%F), was calculated by dividing the dose-normalized oral plasma AUC1 with the dose normalized historical parenteral plasma AUC1 for ISIS 104838 when dosed as a bolus s.c. injection at a dose of 25 mg16 using the following equation: Relative Plasma BAV ¼
AUCpo =Dosepo 100% AUCsc =Dosesc
Descriptive summary statistics were calculated for all subjects in each arm and for only those subjects for whom gastric emptying time occurred before 16 h post-administration as determined by gamma scintigraphy for Treatment A and by evidence of late absorbing drug (24 h) in Treatments B–E.
Statistical Analysis Descriptive statistics were calculated using standard methodology (Microsoft Excel). Differences in the pharmacokinetic parameters by formulation were tested using a nonparametric one-way ANOVA on ranks (Kruskal–Wallis) using SigmaStat (Version 2.0, SPSS, Inc., Chicago, IL).
study drug was well tolerated, with very few adverse events being reported. Headache and neck stiffness were the most common adverse events, both being reported by only two subjects. Review of laboratory data during the course of the study showed no abnormalities that were considered to be attributable to study medication. Figure 3 presents the gastric emptying time for each of the five capsules of ISIS 104838/C10 following administration of Treatment A. These data indicate that in the majority of subjects, under fasting conditions, the capsules were emptied from the stomach within 5 h. In four subjects (#8, 10, 12, and 15) a number of the capsules exhibited gastric emptying times greater than 16 h. Individual ISIS 104838 plasma concentration/time profiles for Treatment A are presented in Figure 4. When there was evidence of minitablets releasing after the predefined pharmacokinetic sampling scheme (gastric emptying time occurring >16 h post-administration), we observed Cmax values of <0.1 mg/mL and/or measurable drug concentrations at 24 h. On this basis pharmacokinetic data for these four subjects have been redacted from calculation of treatment averages. Gamma scintigraphy was not available for Treatment groups B through E. However, evidence of delayed gastric emptying can be inferred from pharmacokinetic profiles that exhibit measurable drug levels 24 h after oral administration, since no subject that exhibited early gastric emptying exhibited measurable plasma drug levels at 24 h. Mean plasma concentration–time profiles and descriptive statistical summary pharmacokinetic
RESULTS Fifteen subjects were enrolled into the study with different numbers completing each treatment. For Treatments A, N ¼ 15; for B through D, N ¼ 14; for Treatment E, N ¼ 12. None of the enrolled subjects had any significant findings in their medical histories or upon physical examination upon screening. One subject failed to return to the clinic following Treatment C (second visit: Subject No. 1), while two additional subjects (Subject Nos. 3 and 15) were lost to follow up in the interim period between Treatments D and E. Overall the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
Figure 3. Gastric emptying time for each of five capsules (single dose) by subject as determined by gamma scintigraphic tracking of the samarium radioisotope. Each diamond represents one capsule—Treatment A. DOI 10.1002/jps
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Figure 5. Mean ISIS 104838 concentration–time profiles in plasma following oral administration of various test formulations. Each data point represents the mean of 6–13 individual measurements and the error bars are standard deviation.
Figure 4. Spaghetti plots of ISIS 104836 plasma concentration–time profiles following administration of Treatment A.
parameters for ISIS 104838 by treatment are shown in Figure 5 and Table 3, respectively. Under fasted conditions mean absorption lag time (Tlag) and time to maximum plasma concentration (Tmax) were similar for all formulations. When administered following a high fat meal, a significant delay was observed in both these parameters compared to that under fasted conditions, although no significant differences were observed in the parameters Cmax or AUC. The plasma BAV relative to parenteral administration of ISIS 104838 by subject and treatment arm is presented in Table 4. Individual subject BAV of ISIS 104838 was highly variable and ranged from 0.7% to 27.5% across the entire data set when the formulations were administered following an overnight fast and exhibited complete stomach emptying prior to 16 h. Only 6 of 12 dosed subjects in Treatment E (post-fatty breakfast) exhibited evidence of complete stomach emptying in a manner that allowed for pharmaDOI 10.1002/jps
cokinetic assessment. For these six subjects, Cmax and AUC were similar to fasted state. Overall, two subjects were identified that exhibited a consistent pattern of delayed gastric emptying and subsequent capsule disintegration (Subjects #10 and 15) confirmed by both scintigraphy (Treatment A) and late (24 h) absorbing ASO (Treatments B–E). Assessment of the effect of gastric emptying time and anatomical site of disintegration on relative bioavailability in Treatment A is presented in graphic form (Figs. 6 and 7). There was no statistically significant effect for either parameter. However, there was a trend for decreased bioavailability with greater delay in gastric emptying as well as disintegration that occurred more distal to the stomach.
DISCUSSION Development of a successful formulation approach for delivery of ASOs orally is expected to be applicable across the technology platform. The applicability of the formulations across the platform derives from the fact that the pharmacokinetics of ASOs are similar across multiple ASO compounds independent of sequence differences and thus highly dependent upon ASO chemisJOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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Table 3. Average Pharmacokinetic Parameters by Treatment Treatment Pharmacokinetic Parameter N (sample number) ISIS 104838 Dose (mg) Tlag (h) Tmax (h) Cmax (mg/mL) AUC1 (mg h/mL) %Bioavailabilityc
A
B
C
D
Ea
11 500 1.56 (0.22) 3.33 (0.20) 1.80 (0.35) 3.28 (0.60) 10.1 (1.96)
13 500 1.23 (0.27) 3.50 (0.27) 1.63 (0.22) 2.91 (0.40) 8.72 (1.20)
12 500 1.33 (0.14) 3.13 (0.31) 1.29 (0.29) 2.39 (0.64) 7.16 (1.93)
10 700 1.15 (0.15) 2.90 (0.46) 2.88 (0.61) 5.60 (1.30) 12.0 (2.78)
6 700 4.25 (0.54)b 6.75 (1.33)b 2.27 (0.64) 6.01 (1.42) 12.9 (3.93)
N, number of subjects included in average. Each average is comprised of 6–13 individuals. Each pharmacokinetic parameter estimate is presented as the average (standard error). a Post-prandial dosing, 2 h following a fatty breakfast. b Significantly delayed ( p < 0.05). c Relative bioavailability as compared to a subcutaneous dose of 25 mg ISIS 104838.
try.21 The development of the second generation chemistries, notably the 20 -O-(2-methoxyethyl) modification in the nucleosides at each of the five terminal 30 and 50 nucleotide sugars of PS oligonucleotides has led to compounds which have an increased binding affinity for the target mRNA and resist endogenous exonucleases thereby prolonging drug half-life.5,16 ISIS 104838 is an example of this new class of modified phosphor-
othioate ASOs involving partial 20 -alkoxy modifications of the ribose sugars in the backbone of the oligonucleotide. These initial results in human volunteers provide guidance for further oral development applicable to other ASOs in this chemical class. The prototype oral formulations presented in this study included two primary components: (1) enteric coating of the filled capsule and (2) a
Table 4. Relative Plasma Bioavailability (%) Summary by Subject (Male Volunteers) and Treatment (Formulation) Treatment Subject# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Average standard deviation (all) Average standard deviationb
A
B
C
D
Ea
13.9 3.51 13.0 8.83 1.72 4.90 21.5 1.89 16.2 0.02 1.70 7.13 11.5 14.0 1.82 8.11 6.59 10.1 6.49
Not dosed 4.12 3.97 15.1 5.85 5.42 14.0 9.03 2.16 12.7 6.95 10.8 14.1 9.35 1.29 8.19 4.62 8.72 4.34
6.28 0.67 4.23 10.8 7.53 1.06 25.9 6.75 0.22 0.73 2.99 6.08 3.69 9.93 Not dosed 6.21 6.62 7.16 6.69
Not dosed 1.96 5.39 3.88 13.9 1.89 18.8 27.5 2.01 1.20 3.11 18.8 19.1 7.63 1.45 9.04 8.78 12.0 8.78
Not dosed 0.03 Not dosed 0.08 1.73 12.40 28.1 0.65 1.30 1.28 4.73 15.6 15.8 3.01 Not dosed 7.05 8.92 12.9 9.62
Treatment A, novel rapid IR minitablets; Treatment B, pulsatile minitablets (50/50 rapid pulse); Treatment C, pulsatile minitablets (50/50 slow pulse); Treatment D, pulsatile minitablets (70/30 rapid pulse); Treatment E, pulsatile minitablets (70/30 rapid pulse) dosed 2 h after fatty breakfast. a Fatty breakfast fed arm (2 h post-prandial dosing), all other arms were fasted overnight. b Redacted average ¼ removed AUCs () for which gastric emptying of enteric capsules occurred after 16 h post-administration) as determined by gamma scintigraphy for Treatment A and by evidence of late absorbing drug (24 h) in Treatments B–E. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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Figure 6. Bioavailability appears to decrease as the ‘‘delta GE’’ (measured by determining the difference in time between first and last capsule leaving the stomach) increases beyond 1 h. Analysis is not powered to provide statistical significance. Each symbol represents one subject. Subjects 8, 10, 12, and 15 were not included in this analysis (some capsules did not leave stomach during the scintigraphic measurement period) since plasma profiles are not available for the late emptying capsules.
series of ‘pulsatile release’ dosage forms designed to give immediate release of the oligonucleotide concurrent with a measured amount of C10 (true for all formulations) followed by a second pulsed
Figure 7. Anatomical position for Treatment A capsule disintegration as determined by gamma scintigraphy and its effect on plasma bioavailability of ISIS 104838. Each point represents one subject. Subjects 8, 10, 12, and 15 were not included in the analysis since scintigraphy data was not available for late gastric emptying capsules. DOI 10.1002/jps
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release of the balance of C10 component (Formulations B, C, and D). The requirement of the enteric coat relates to the properties of both the oligonucleotide and the permeation enhancing excipient, C10, in that ISIS 104838 and other oligonucleotides of similar chemistry are susceptible to acid catalyzed depurination while C10 is converted to its unionized acid form which is an insoluble oil at 378C. The pulsatile release component incorporated in these prototype formulations—immediate release (IR) followed by a delayed release (DR)—maximizes oligonucleotide uptake by prolonging the luminal presence of the C10 component at concentrations relating to permeation action. In effect, the two pulses of C10 sustain, for a brief duration, the C10 luminal concentrations as it is rapidly cleared from the small intestine by both dilution and absorption mechanisms. Putatively, this longer C10 residence time within the gut lumen allows for a prolonged duration of membrane permeability and extended mucosal surface area for the oligonucleotide to be absorbed. This formulation approach is slightly different from the synchronous release of C10 and drug that has been proposed in the literature by Baluom et al.22,23 In contrast, the formulations tested in this study are designed to: initially establish high concentrations of both oligonucleotide and C10 followed by a continued or extended release of supplemental C10. This design takes advantage of the firstorder absorption properties of oligonucleotides which are optimized with high concentrations of oligonucleotide in the presence of active concentrations of C10. Put another way, the oligonucleotide absorption process is far more effective if all of the oligonucleotide is in solution and at high luminal concentration during the brief window available for absorption—since the rate of absorption would be dependent on the oligonucleotide concentration (data not shown). As described above, all four capsule formulations tested in this study had an immediate releasing minitablet population resulting in a fast release of the oligonucleotide and C10. One of these formulations (Formulation A) only had this IR minitablet population and therefore served as a ‘‘single pulse’’ control for the remaining formulations. Among the three formulations having delayed releasing (DR) C10 minitablets, the second pulse of C10 was designed to have a delay of either 20 or 40 min with the 20 min delay termed as a ‘‘rapid pulsatile’’ and the 40 min delay termed ‘‘slow pulsatile’’. It’s important to note JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
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here that after minitablet encapsulation and enteric coating of the intact capsule it is clear that an additional lag time is evident as seen in Figure 2 where the 40 min C10 release was not complete until approximately 60 min. The results of the study suggest that the ‘‘rapid’’ C10 pulse release may provide some improvement in overall systemic bioavailability (Formulation D; used in Treatments D and E) wherein 70% of the C10 and 100% of the oligonucleotide is released in the IR portion followed by the remaining 30% of the C10. While the trend for improvement compared to the single IR formulation (A) and the two other two-pulse formulations (B and C) does exist, the increase is small and not statistically significant. Pharmacokinetic studies of the 20 -O-(2-methoxyethyl) modified PS oligonucleotides in both animals and man following intravenous and subcutaneous administration have led to the following observations: (1) the plasma pharmacokinetics of ISIS 104838 following parenteral administration is descriptive of distribution; plasma clearance being dominated by tissue distribution,5 (2) the plasma pharmacokinetics are somewhat nonlinear with increasing doses exhibiting somewhat greater than dose proportional increases in AUC and with more rapid distribution at doses of 0.5 mg/kg or less,16 (3) they are slowly cleared from tissue by relatively slow nuclease metabolism and efflux into plasma followed by urinary excretion of the shortened metabolite products, and (4) S.C. administration results in essentially complete absorption of oligonucleotide.5 The known nonlinear distribution component of oligonucleotide pharmacokinetics was factored into the selection of an appropriate reference parenteral dose. The historical (external control) parenteral dose selected (25 mg) represents 5% of the delivered oral dose and produces similar plasma concentrations to those observed following oral administration at a dose of 500 mg. Parenteral pharmacokinetics of oligonucleotides in general and specifically for ISIS 104838 is well behaved with reproducibility across studies and without observed differences between normal volunteers and patients in numerous clinical studies (unpublished comparisons, ISIS Pharmaceuticals, Inc.). Thus, with the primary goal of this study being the comparison of numerous formulation prototypes, an external control at an exposure-equivalent dose administered subcutaneously (s.c.) in accordance with current clinical practice for ISIS 104838, and JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 1, JANUARY 2008
other ASOs in this chemical class, was deemed appropriate. The use of gamma-scintigraphy allowed visualization of the movement of the minitablets throughout the GI tract of the male volunteers and permitted evaluation of gastric emptying and gastrointestinal transit on the performance of the IR ASO formulation. Using these data we were able to correlate the observed delayed gastric emptying of the minitablets in a subset of subjects with late absorbing drug, which allowed for the identification of delayed gastric emptying subjects based on pharmacokinetics alone for the remaining prototypes that were tested without scintigraphy. The reduced apparent bioavailability observed for late gastric emptying of capsules is likely an artifact of the pharmacokinetic sampling schedule such that any oligonucleotide released from the capsules and absorbed after sampling is complete would not be included in the measured AUC. In addition to identification of late gastric emptying subjects, the gammascintigraphy data also provide confidence that oligonucleotide was absorbed from all regions of the small intestine without significant limitations. While there was an apparent trend for lower absorption in the distal small intestine compared with proximal small intestine, the number of observations does not provide appropriate power and conclusions in this regard should be taken with caution. Plasma concentration data taken after oral administration tends to underestimate the endorgan bioavailability of ISIS 104838.14 In a study in dogs, Raoof et al.14 determined the tolerability and pharmacokinetics of ISIS 104838 following multi-dose administration by intravenous and oral routes. In this dog study, an enteric-coated tablet formulation of ISIS 104838 (80 mg) and C10 (330 mg) was administered by oral gavage daily for five days. The average plasma BAV, based on the ratio of plasma AUC Oral: IV averaged 1.4%. The tissue distribution profile was similar following either route of administration, and the mean systemic target tissue BAV ranging from 2.0% to 4.3%, relative to tissues following IV administration. Distribution was dependent on the tissue type with the highest concentrations of intact oligonucleotide achieved in the kidney and liver. These organs demonstrated the highest and most consistent bioavailability as well. For antisense drugs, such as ISIS 104838, the tissue bioavailability determines pharmacological activity 24 since the target is intracellular mRNA. DOI 10.1002/jps
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Based on the nonclinical studies discussed above, it seems reasonable to posit that the average relative plasma bioavailability of 7–13% attained in this study with the various formulations represents an underestimate of the actual tissue bioavailability of ISIS 104838. While the effective plasma distribution half-life of 20 -O-(2methoxyethyl) modified PS oligonucleotides is approximately 1–2 h, for ISIS 104838 a prolonged phase with a terminal half-life of about 27 days has been identified16 that likely reflects the return of ISIS 104838 from tissue stores to plasma where it is then rapidly eliminated. This suggests that tissue accumulation upon repeat daily administration will be substantial. These two properties, the underestimation of true tissue bioavailability from plasma data and the prolonged tissue halflife of ISIS 104838, provide good confidence that these prototype formulations will deliver active amounts of ASO to the target tissues. The long half-life also mitigates variability in bioavailability. The high degree of variability observed in this study is substantial. We hypothesize that the observed variability in bioavailability within and between subjects will be reflected in routine multiple dose administration. When modeled using first-order pharmacokinetic models, integrating a random bioavailability from day to day, the accumulation in tissue at steady-state reflected the average bioavailability over a period of several months. In other words, variability in daily bioavailability was mitigated by the long half-life yielding ultimate tissue levels that are predicted to be relatively stable at steady-state. In conclusion, the combination of an absorption enhancer (sodium caprate), the 20 -O-(2-methoxyethyl) modified PS oligonucleotide (ISIS 104838) in a multiparticulate, minitablet presentation, and delivered within enteric coated gelatin capsules yielded measurable plasma concentrations for ISIS 104838 in all healthy male volunteer subjects. Based on the current study, oral dosing of ISIS 104838 results in measurable plasma bioavailability of at least 10% relative to subcutaneous administration. It is likely that the plasma bioavailability underestimates the true tissue bioavailability of this compound based on preclinical evaluations. These data taken together with our previous preclinical experience provide confidence that clinical oral application would be successful for modified ASOs. The value of this technology is clear when one considers that positive pharmacology of this class of oligonucleotide is demonstrated with dosing regimens as low DOI 10.1002/jps
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or lower than 200 mg administered weekly by the subcutaneous route.25 With oral dosing of about 400 mg daily—from four capsules as formulated in this study where approximately 10% bioavailability would be expected—the systemic exposure should exceed that required for therapeutic efficacy.
ACKNOWLEDGMENTS This work was supported by Orasense Ltd., a joint venture between ISIS Pharmaceuticals, Inc. and Elan Pharmaceuticals. As of JUN2004, Orasense is now a wholly owned subsidiary of ISIS Pharmaceuticals, Inc. We thank Dr. Tony Fitzpatrick and Triona Healy of Elan Pharmaceuticals for management and oversight of this trial and Dr. A.L. Connor and Dr. Andrew Salmon, for their assistance in the scientific and medical conduct of the study at PPL. We thank Dr. Rosie Yu, Dr. Arthur Levin and Dr. Stanley Crooke for their expert review and helpful discussions. Special thanks to Robert Saunders for his expert assistance in the preparation of this manuscript.
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