pharmacokinetics of oral cyanocobalamin formulated with...
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Clinical Therapeutics/Volume 33, Number 7, 2011
Pharmacokinetics of Oral Cyanocobalamin Formulated WithSodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC): AnOpen-Label, Randomized, Single-Dose, Parallel-Group Studyin Healthy Male Subjects
M. Cristina Castelli, PhD; Diane F. Wong, MS; Kristen Friedman, MPh; andM. Gary I. Riley, DVM, PhD
Emisphere Technologies, Cedar Knolls, New Jersey
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ABSTRACTBackground: Vitamin B12 (cobalamin) deficiency
may be caused by inadequate dietary intake of B12 orby conditions that result in malabsorption of the vita-min. Crystalline vitamin B12, usually in the form ofcyanocobalamin, is administered parenterally (ie, in-tramuscularly) or orally for treating deficiency states.Intramuscular administration is widely accepted as atreatment method. Oral B12 supplementation is alsoused, but it is considered to be less reliable.
Objective: This study was conducted to compare theharmacokinetics and tolerability of 2 oral formula-ions of cyanocobalamin—a marketed cyanocobala-in tablet (immediate-release B12 5 mg) and cyanoco-alamin formulated with a proprietary carrier, sodium-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC)—to es-
ablish the feasibility of using an absorption enhancerith B12 to improve uptake of the vitamin. This was
he first clinical study conducted with the cyanocobal-min/SNAC coformulation.
Methods: An open-label, randomized, single-dose,arallel-group study was conducted in healthy maleubjects. Subjects were randomly assigned to 1 of 4reatment groups: Treatment A subjects (n � 4) re-eived 2 tablets of 5-mg cyanocobalamin formulatedith 100-mg SNAC as part of a dose range–findingrm included to determine a dose to provide a measur-ble concentration of vitamin B12 at all time pointshen tested with the available vitamin B12 assay;
reatment B subjects (n � 6) received 1 tablet of 5-mgyanocobalamin formulated with 100-mg SNAC;reatment C subjects (n � 6) received 1 commerciallyvailable 5-mg cyanocobalamin tablet; and treatment
subjects (n � 4) received commercially available-mg cyanocobalamin IV. Treatment A was completedweeks before treatments B, C, and D were studied.
uman serum B12 was analyzed by chemilumines-934
ence assay method. Validation procedures establishedhat samples could be diluted up to 100 times withoutny effects on accuracy and precision. The pharmaco-inetic properties of vitamin B12 were characterizedy noncompartmental analysis. Vitamin B12 absoluteioavailability estimates were calculated between theral (A, B, and C) and IV (D) treatments using non–aseline-adjusted vitamin B12 concentrations as wells baseline-adjusted vitamin B12 concentrations, withr without body weight adjustments. Tolerability wasvaluated through review or monitoring of medicalistory, physical examination findings, concomitantedications, vital signs, laboratory tests (hematology,
erum chemistry, and urinalysis values), electrocardi-graphy, adverse events, and serious adverse events.
Results: Twenty healthy male subjects, aged 20 to5 years, participated in this study. Based on data fromreatment A, a 5-mg cyanocobalamin dose was selectedor use with treatments B and C. The oral cyanocobal-min formulation containing SNAC had greater meanbsolute bioavailability than the commercial oral for-ulation (5.09% vs 2.16%, respectively), calculatedn AUC0–last values uncorrected for baseline, weight,r body mass index. It also had a reduced Tmax com-ared with the commercial formulation (0.5 hours vs.83 hours, respectively). The Ke was similar betweenreatments (0.028 1/h vs 0.025 1/h). Comparable re-ults were achieved using corrected values. The cyano-obalamin/SNAC formulation was well tolerated, andhere were no reported adverse events.
Conclusions: An oral formulation of 5-mg cyanoco-alamin containing 100-mg SNAC, an absorption en-
Accepted for publication May 24, 2011.doi:10.1016/j.clinthera.2011.05.0880149-2918/$ - see front matter
© 2011 Elsevier HS Journals, Inc. All rights reserved.
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hancer, provided significantly improved bioavailabilityand a significant decrease in Tmax for B12 in a smalltudy of normal healthy subjects compared with aommercially available 5-mg cyanocobalamin oral for-ulation. Both oral formulations and commercial-mg cyanocobalamin IV were well tolerated. Clinical-rials.gov identifier: NCT01311739. (Clin Ther.011;33:934–945) © 2011 Elsevier HS Journals, Inc.ll rights reserved.Key words: B12, cobalamin, cyanocobalamin, oral,
harmacokinetics, SNAC.
INTRODUCTIONVitamin B12 deficiency is a clinically important condi-tion that occurs when vitamin stores are depletedthrough inadequate dietary intake or impaired vitaminabsorption. Vitamin B12 is most abundant in foods ofanimal origin; therefore, vegetarian diets predispose toB12 deficiency.1 Defective absorption is generally at-tributable to a specific failure of extraction and trans-port of B12 from dietary sources, as, for example, withgastric achlorhydria and inadequate intrinsic factorproduction, or to more generalized disturbances ofgastrointestinal structure and function, such as gastricresection, ileal resection, Crohn’s disease, and bacterialovergrowth of the intestine.
Supplemental vitamin B12, in the form of crystallinecobalamin (common forms include cyanocobalamin,methylcobalamin, and hydroxycobalamin), is admin-istered either parenterally (ie, intramuscularly) ororally to treat vitamin B12 deficiency. Unlike dietaryB12, which is protein bound and requires pepsin andacid conditions in the stomach for release and subse-quent binding to intrinsic factor,2 crystalline cobala-min exists in the free unbound state. Crystalline co-balamins are known to be absorbed by passivediffusion to the extent of �1% over a range of �100�g to 5 mg.3 However, dietary B12 is absorbedthrough the action of intrinsic factor and its receptors,a mechanism that normally limits B12 uptake to be-tween 5 and 10 �g/d.4 Intramuscular administration iswidely accepted as a method of treating B12 deficiency.Oral B12 supplementation, which originated in the1960s, continues to be studied.3 An area of interest inhe use of oral supplements has been in the elderly, inhom food-cobalamin malabsorption becomes in-
reasingly prevalent after 50 years of age.5 In recent
ears, doses of 1000 to 2000 �g/d have been used inJuly 2011
clinical studies to restore B12-deficient patients to thenormal range. Successful treatment of patients withB12 deficiency has been observed also with lowerdoses, but the proportion of patients respondingdecreases.6,7
An earlier preclinical study in rats8 reported that thextent of vitamin B12 absorption was significantlyP � 0.05) enhanced when cyanocobalamin was ad-inistered in combination with the absorption enhancer
odium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC).he present 4-arm, open-label, randomized, single-ose, parallel-group study investigated the pharmaco-inetic profile of a new formulation* of cyanocobala-in containing SNAC in 20 healthy males. SNAC haseen tested in nonclinical9,10 and clinical studies as a
drug delivery agent at dose multiples exceeding thoseused for cyanocobalamin delivery.11 The 2 ingredientsn this new formulation, cyanocobalamin and SNAC,re both designated generally recognized as safeGRAS) according to US Food and Drug Administra-ion regulatory definitions pertaining to food and fooddditives. In both cases this status was granted inde-endently of the present clinical study.
SUBJECTS AND METHODSStudy Design
This was an open-label, randomized, single-dose,parallel-group pharmacokinetic (PK) study conductedat a single center (MDS Pharma Services, Neptune,New Jersey) between May 22, 2008 and June 16,2008. Our objective was to assess the PK profile andtolerability of oral cyanocobalamin coformulated withSNAC and a commercial 5-mg, immediate-release for-mulation† of cyanocobalamin. The formulations areidentified as cyanocobalamin/SNAC and commercialcyanocobalamin. The study was approved by the inde-pendently functioning institutional review board (IRB)of MDS Pharma Services and was conducted in com-pliance with Section 56 of Title 21 of the Code ofFederal Regulations (CFR) and in accordance with theclinical research guidelines established by the MedicalResearch Council of Canada, the basic principles of theDeclaration of Helsinki defined in US 21 CFR Part312.20, the requirements of Directive 2001/20/EC
*Trademark: Eligen B12® (Emisphere Technologies, Inc, CedarKnolls, New Jersey).
†Trademark: Vitalabs® Immediate Release B-12 (Vitalabs, Inc,
Jonesboro, Georgia).935
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(Europe), the principles enunciated in the Declarationof Helsinki (Edinburgh, 2000), and the ICH Harmon-ised Tripartite Guideline: Guideline for Good ClinicalPractice.
The selected doses of 10 mg (2 � 5-mg cyanocobal-amin/100-mg SNAC oral dose) and 1 mg (IV dose)were expected to yield B12 concentrations exceedingthe endogenous baseline and consequently to allow fullPK assessments. The design of the study allowed for adecrease in the oral dose of cyanocobalamin if, follow-ing the pilot part of the study (treatment A, conducted3 weeks before the remaining treatments), it was deter-mined that relevant concentrations for PK analysiscould be achieved following a 5-mg dose.
SubjectsRecruitment was conducted using the MDS Pharma
Services database. Subjects were called and askedwhether they wanted to participate in the study. Dur-ing enrollment subjects were asked to read, under-stand, and sign the IRB-approved informed consentform (ICF). In addition to elucidating the purpose andconduct of the study, the ICF clearly stated the sub-jects’ responsibilities, which included following gen-eral clinic rules, following study instructions given bythe staff, following study restrictions, reporting anychanges in physical or mental condition during thestudy, reporting any side effects experienced during thestudy, and giving true and complete answers to ques-tions during the study. Subjects were compensated,and payment was prorated at the completion of eachperiod (completion of confinement for dosing/sam-pling and completion of study). Subjects assigned to IVdosing were paid slightly more than subjects assignedto oral dosing.
Healthy male subjects aged 18 to 45 who weighedbetween 60 and 100 kg and had a body mass index(BMI) between 18 and 30 kg/m2 were eligible to receivereatments A, B, C, or D, as per inclusion criteria, ifhey had normal organ function, including renal andepatic function; normal vital signs and electrocardi-graphy (ECG) results; normal results on routine he-atology and clinical chemistry tests; and normal lev-
ls of serum B12 (193–982 pg/mL), methylmaloniccid (0.0–0.4 �mol/L), and homocysteine (5–12 �mol/
L). Exclusion criteria included current use of acetamin-ophen or nonsteroidal antiinflammatory drugs, antibi-
otics, antacids, multivitamins, or nutritional supp-936
lements and absolute platelet count below 100 �109/L.
Eligible subjects entered the clinic the evening beforestudy product administration (check-in) and remainedin the clinic for at least 24 hours postdose (2-nightconfinement) until the conclusion of the treatment andassessments. Subjects were discharged after the lastblood sample was taken and the appropriate safetyparameters (eg, vital signs, ECG) remained within ac-ceptable ranges. Clinical laboratory tests, urine drugscreen, and urine alcohol test were performed prior todosing. While in the clinic, all subjects began a 10-hourovernight fast prior to dosing. Vital signs and baselinePK blood samples were taken within 30 minutes beforestudy product administration. Vital signs were taken,adverse event (AE) monitoring was performed, andblood samples were drawn for up to 24 hours afterstudy product administration to measure the PK prop-erties of cyanocobalamin and obtain tolerability data.Standard meals were given for the duration of confine-ment beginning at 4 hours postdose.
Test FormulationsThe study product used for treatments A and B was
a cyanocobalamin/SNAC tablet containing 5-mg cya-nocobalamin and 100-mg SNAC (Emisphere Technol-ogies, Inc, Cedar Knolls, New Jersey; lot no. 112-08-01, expiration date April 23, 2009). The comparatorproduct was a commercial 5-mg, immediate-release cy-anocobalamin tablet (Vitalabs, Inc, Jonesboro, Geor-gia; lot no. 22083, expiration date March 2010). TheIV product was a commercial 1-mg cyanocobalaminsolution (1 mg/mL) (American Regent, Inc, Shirley,New York; lot no. 8071, expiration date January2010).
TreatmentsTreatment A was a single oral dose of cyanocobal-
amin/SNAC administered to 4 human subjects in thefasted state as 2 tablets taken with 50 mL of water fora total dose of 10-mg cyanocobalamin and 200-mgSNAC. Treatment A was administered as a screeningstep to enable selection of a dose resulting in an in-crease in serum B12 concentrations that would be mea-surable above the baseline value for a period of 24hours and be suitable for PK evaluation. This doserange–finding arm was completed 3 weeks before
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Treatment B, the test treatment, was a single oraldose of 5-mg cyanocobalamin/100-mg SNAC admin-istered in the fasted state to 6 subjects as 1 tablet takenwith 50 mL of water.
Treatment C, the comparator treatment, was a sin-gle oral dose of commercial 5-mg cyanocobalaminalone administered in the fasted state to 6 subjects as 1tablet taken with 50 mL of water.
Treatment D, the reference treatment, was a single1-mg IV dose of commercial cyanocobalamin admin-istered in the fasted state to 4 subjects. Each subjectreceived a 1-mL IV injection of a 1-mg/mL solution,resulting in a total dose of 1-mg cyanocobalamin.
Blood Sample ProcessingFollowing each treatment, A, B, C, or D, 25 blood
samples were drawn by venipuncture for B12 analysiswithin 30 minutes predose and at minutes 2, 5, 10, 20,30, 40, and 50 and hours 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 14, 16, 20, and 24 postdose. Five mL of wholeblood were collected into a prelabeled 5-mL red top orserum separator tube (SST tube) at each sampling timepoint under fluorescent lighting, repeatedly inverted asper manufacturer’s instructions and allowed to clot for30 minutes prior to centrifugation at 3000 rpm for 7minutes at 5°C. The samples were removed from thecentrifuge under yellow light and transferred to 2 1-mLtransfer tubes prior to placement in a freezer at –20°C.Approximately 2 mL of serum was collected per sam-ple. Each serum sample was split in half (�1 mL peraliquot). One duplicate sample was shipped to MDSBioanalytical Laboratories (Lincoln, Nebraska) forB12 analysis and the other was retained as a backupsample. Sampling in subjects who received the IV dosewas performed in the arm not used for dosing.
Sample AnalysisThe B12 assay used in this study to analyze samples
following treatment A, B, C, or D is a competitive mul-tistep assay that involves an alkaline denaturation ofendogenous binding proteins using a dithiothreitol(DTT) and a sodium hydroxide/potassium cyanide(NaOH/KCN) solution and immunoreaction steps,during which the released vitamin B12 from testsamples competes with immobilized vitamin B12 forbinding to hog intrinsic factor. The Immulite 2000analyzer and vitamin B12 reagent kit used were pur-chased from DPC (now Siemens Medical Solutions,
Flanders, New Jersey). tJuly 2011
The following steps were followed to operate theinstrument as described in the Immulite 2000 opera-tors manual (document no. 600149-03-D), Section 6(Routine Operations)12: (1) Calibrate the vitamin B12channel of the Immulite 2000 using the low and highadjustors (included in the DPC Immulite 2000 VitaminB12 Reagent Kit; used as described in the package in-sert PIL2KVB-22, 2006-04-1813); (2) Transfer at least350 �L of each standard, quality control (QC), andnknown patient samples into appropriate samplingubes to be loaded onto the instrument; (3) Place eachample tube on the instrument in the same order as theunsheet; (4) Program the instrument to analyze eachample for Vitamin B12. The assay was performed andalidated by MDS Pharma Services (report on file).14
During standard kit validation, a set of calibration ver-ifiers (160–1120 pg/mL) were run at the beginning ofeach curve, along with at least 6 replicates of QC samplesat 3 concentrations (178, 423, and 799 pg/mL or 178,423, and 1012 pg/mL). The 5 calibration standard sets(160–1120 pg/mL) assayed over a period of 4 days deter-mined the interday and intraday reproducibility, alongwith benchtop stability, freeze/thaw stability, and refrig-eration stability. Long-term storage stability at –20°Cwas determined at 178, 423, and 799 pg/mL. Due to thehigher than expected vitamin B12 in clinical samples, anew calibration standard range was necessary. Therefore,for V2 (update) validation, VB12_V09 was run on July17, 2008 to validate new standard 5 (1120 pg/mL) andnew high QC (1012 pg/mL). During the validation, QCsamples were stored in a freezer set at –20°C.
Before validation, the vitamin B12 channel of the Im-mulite analyzer was calibrated. Two non-zero adjustorswere used to correlate the counts per second (cps) of theinstrument’s electronically stored master standard. Theanalyte concentration is determined from that adjusted2-point curve, as it relates to the master standard curve.From these 2 known values, the instrument established aresponse factor that it used to calculate the concentrationof vitamin B12 in any given samples using the immuno-metric (competitive) assay equation:
CPS � P2 �P1 � P2
P4
1 � �Dose ⁄ P3�where P1 is maximum cps, P2 is minimum cps (NSB);
3 is dose at half the maximum cps, and P4 is slope of
he logit-log plot.937
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The vitamin B12 sample concentrations were thenprinted out on the instrument’s printer. The protein-based verifiers prepared for this validation were usedonly to verify the linear range of the assay, with nostandard curve generated. The following performanceparameters were validated and documented in the val-idation report: specificity, quantification limit, calibra-tion range, intraday and interday accuracy and preci-sion, dilutional linearity, and stability (refrigerations,on-system, benchtop, freeze/thaw, and long-term).
Percent recoveries at all calibration verifier levelswere between 97.1% and 113.5%, and the relativestandard deviation (%RSD) was between 2.4% and9.6%. All verifiers were within acceptance criteria. Pre-cision and accuracy was evaluated by analyzing 4 rep-licates of 3 concentrations of the QC verifiers on thesame day (intraday accuracy and precision) or on dif-ferent days (interday accuracy and precision). The 3concentrations were 178, 423, and 799 pg/mL. Intra-day and interday accuracy and precision were verifiedwithin the serum vitamin B12 concentration range of160 to 1120 pg/mL. The percent recoveries and %RSDfor replicates at each level were within acceptance cri-teria. Samples could therefore be diluted up to 100times without any effects on accuracy and precision.The sample with a target value of 83,727 pg/mL (�100dilution) showed a lack of accuracy (124.8% recov-ery). This higher than expected recovery appeared to
Table I. Vitamin B12 method validation: dilution line
Target Values (pg/mL) 2806 4881
DF 5 10Result 1 2787 5034Result 2 3174 5059Result 3 2740 5121Result 4 2794 4738Result 5 3066 4651Result 6 2767 4947Mean 2888 4925SD 184 189%RSD 6.4 3.8Mean % Recovery 102.9 100.9N 6 6
DF � degrees of freedom; RSD � relative standard deviatio
be sample related, rather than dilution factor related,
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as the sample with a target value of 42,234 pg/mL andthe same dilution factor (100) did show acceptable ac-curacy and precision. Table I summarizes the resultsor dilutional linearity.
Pharmacokinetic AnalysisThe PK properties of B12 were characterized by
noncompartmental analysis using WinNonlin Profes-sional 5.2 (Pharsight, St. Louis, Missouri). The nomi-nal PK sampling times were used for PK analysis unlessthe actual sampling times deviated significantly fromthe nominal time points according to the statisticalanalysis plan. Serum PK parameters determined forB12 included Cmax, Tmax, AUC0–last, AUC0–�, t1/2, andKe.
15 Serum B12 PK parameters were also calculatedsing baseline-adjusted B12 values. Baseline-adjustedndividual serum B12 concentrations were obtained byubtracting the predose concentration from postdoseoncentration at each time point. Baseline-adjusted PKnalysis was performed based on baseline-adjusted se-um B12 concentration–time data. Slightly negativealues obtained following baseline adjustment werereated as zero for the PK analysis. Ke and other rele-
vant PK parameters (t1/2 and AUC) were not estimatedin cases in which the terminal phase of the log-concen-tration–time profile exhibited a linear decline phasewith regression coefficients �0.85. At least 3 or moredata points (excluding Cmax) in the terminal phase
results.
9032 9032 42,234 83,737
20 40 100 10042 8553 43,097 107,77427 8599 43,940 113,95543 8204 41,199 106,44677 9210 40,343 100,43488 8294 41,678 100,12005 8481 40,857 98,08397 8557 41,852 104,46922 354 1388 6013
3.6 4.1 3.3 5.899.6 94.7 99.1 124.8
6 6 6 6
arity
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were used for Ke calculations. Baseline cobalamin mea-
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surements were limited to a single sample per subjectbecause high (5-mg) doses of cyanocobalamin wereadministered.16 Circadian changes in B12 do not occurxcept as a possible consequence of plasma volumehanges during bedrest.17 In these circumstances, re-
peated measures of baseline B12 were not considerednecessary. This was confirmed by the study data, whichdemonstrated that baseline corrected Cmax values forB12 greatly exceeded baseline values (mean baselinescreening value for all subjects, 512 [216] pg/mL; meanCmax value for the test formulation, 12,847 [6613] pg/mL; mean Cmax value for the commercial formulation,1239 [450] pg/mL).
B12 absolute bioavailability (%F) estimates were per-formed on AUC0–last values (% of F where F � AUCoral �
DoseIV/AUCIV � Dose oral) between the oral (A, B, and C)nd IV (D) treatment formulations. The AUC0–last pa-ameter was calculated using non–baseline-adjusted cya-ocobalamin concentrations as well as baseline-adjusted12 concentrations, with or without body weightdjustments.
TolerabilityAll subjects were included in the tolerability analy-
sis. Clinical tests for inclusion/exclusion criteria andtolerability were conducted by a CLIA- and CAP- cer-tified laboratory (MDS Pharma Services, Neptune,New Jersey). A responsible physician was present onsite for the duration. A product safety physician withexpertise in the new formulation was on call. Tolera-bility was evaluated through the monitoring or reviewof medical history findings, physical examination find-ings, concomitant medications, vital signs (includingblood pressure, respiratory rate, heart rate, and tem-perature), laboratory tests (hematology, serum chem-istry, and urinalysis values), ECG, AEs, and seriousadverse events. Vital sign measurements (systolicblood pressure, diastolic blood pressure, heart rate,respiration rate, and temperature) were performed inthe sitting position at screening, check-in (day 1, pre-dose), and at approximately 1, 2, 3, 5, 8, 12, 16, and 24(end of study) hours postdose. Clinical laboratory testswere performed at screening, check-in (day 1, pre-dose), and end of study and were evaluated by theinvestigator. ECG was performed at screening andend of study. Physical examinations were conductedat screening, check-in, and end of study. Tolerabilityanalyses were performed and a report was prepared
at MDS Pharma Services, Department of ClinicalJuly 2011
Pharmacology. AEs were coded using the MedicalDictionary for Regulatory Activities (MedDRA)11.0.18 AEs were collected by the nursing stafftrained to address the subject with open-ended ques-tions, such as “How are you feeling this morning?”.If the subject offered a complaint, the nurse inquiredfurther regarding onset, associated symptoms, pre-cipitating or aggravating or relieving factors, andresolution time. Data for AEs were analyzed usingthe treatment-emergent AE (TEAE) philosophy. TE-AEs were defined as AEs that emerged during treat-ment, having been absent at pretreatment, or thatworsened in severity or frequency relative to the pre-treatment state. Predose values were defined as thelast observation obtained prior to day 1 dosing. Shifttables describing out-of-range shifts from predose topostdose were created by treatment group. The shifttables were generated by the project programmer.Another peer programmer and project statisticianreviewed these tables. The medical writer then usedthese tables to write the safety text. The principalinvestigator reviewed and evaluated the original val-ues. Out of normal range and clinically significantlaboratory values were listed by subject. For vitalsigns, descriptive statistics (N, mean, SD, median,minimum, and maximum) were reported for vitalsign measurements and change-from-predose (be-fore dosing of each period) values by time point andtreatment group. For ECG, descriptive statistics (N,mean, SD, median, minimum, and maximum) werereported for ECG measurements (ventricular rate,PR, QRS, QT, and QTc [using Bazett’s formula])and change from screening values by treatmentgroup. ECG results were also classified as normal orabnormal (clinically significant and not clinicallysignificant) and a shift table was created to describeshifts from screening to end of study by treatmentgroup. Individual clinically significant results wereto be discussed. For physical examinations, a shifttable was created to describe shifts from check-in toend of study by treatment group for each body sys-tem examined. All medications, as documented bythe investigator, were coded using the WHO DrugDictionary, October 2007.19 Data listing of priorand concomitant medications were provided. De-scriptive statistics were calculated for continuous de-mographic variables (age, height, weight, and BMI)and frequency counts were tabulated for categorical
demographic variables (race and gender).939
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Statistical MethodsPharmacokinetics
Serum B12 concentrations and PK parameters weresummarized using descriptive statistics (mean, SD,%CV, geometric mean, N, median, minimum, andmaximum). Mean serum B12 concentration–timecurves were presented graphically on a linear scale(with and without SD) and semi-log scale (with andwithout SD). Individual serum B12 concentration–time curves were also graphically presented. A statisti-cal analysis using a 2-sample t test was performed onndividual values to determine the effects of SNAC onral B12 absorption (ie, Cmax, Tmax, and AUC).20 The
significance criterion was set at 0.05. To this purpose,PK parameters from the commercial cyanocobalaminformulation were compared with the cyanocobalamin/SNAC formulation.
TolerabilityTo review the safety profile, summary tables and
change from baseline tables, including descriptive sta-tistics for serum chemistry, hematology, and urinaly-sis, were created. Shift tables describing out-of-rangeshifts from baseline to postdose were also created. AllECG results were summarized using descriptive statis-tics and were classified as normal or abnormal, and ashift table was created to describe shifts from predoseto end of study.
Descriptive statistics for vital sign measurements ateach time point and change from baseline to each timepoint were also created. Physical examination resultswere reviewed by summarizing the data by a shift table,showing shifts in normal/abnormal status fromcheck-in to end of study.
RESULTSDemographic and Other Baseline Characteristics
Twenty healthy male subjects, aged 20 to 45 years,participated in this study. Demographic characteristicsand group mean B12 baseline concentrations areshown in Table II. The mean age was 30 years, themean weight was 76.7 kg (range, 61.3–94.4 kg), andthe mean height was 174 cm (range, 163–180 cm).Body weight was between 60 and 100 kg, and the meanBMI was 25.4 kg/m2 (range, 21.8–30.0 kg/m2). Re-garding race, 15 subjects were black, 3 subjects wereCaucasian, 1 subject was Hispanic, and 1 was Europe-
an/Middle Eastern.940
All subjects satisfied all inclusion and none of theexclusion criteria, with brief verification at check-in(day 1, predose).
Pilot ArmA 10-mg cyanocobalamin/SNAC oral dose (treat-
ment A: 2 � 5-mg cyanocobalamin/100-mg SNAC)administered prior to the other treatments was used asa pilot arm to identify an oral cyanocobalamin dosethat would achieve serum concentrations of B12greater than the endogenous baseline for a full 24-hourPK evaluation. Assuming linear PK properties, the re-sults suggested that a 5-mg dose would be sufficient fortreatments B and C in the treatment period (Table III).
Vitamin B12 PharmacokineticsSerum cobalamin PK parameters following treat-
ments A, B, C, and D are shown in Table III. All valuesre reported as mean (SD). Treatment B had signifi-antly greater Cmax and AUC0–last and a shorter mean
Tmax compared with the treatment C commercial for-mulation (P � 0.01). The same result was observedafter baseline adjustment.
The exposure to 5-mg cyanocobalamin/SNAC(treatment B: 1 tablet of 5-mg cyanocobalamin/100-mg SNAC) and 10-mg cyanocobalamin/SNAC(treatment A: 2 � 5-mg cyanocobalamin/100-mgSNAC) appears to be dose proportional. However, be-cause only 2 dose levels were used in this study and adifferent number of tablets was administered for eachdose, dose proportionality should be confirmed in sub-sequent studies.
The mean serum B12 concentration versus timesemi-log profiles for the 2 5-mg oral formulations and1-mg IV formulation are shown in the Figure. The5-mg cyanocobalamin/SNAC formulation achievedmean Tmax earlier (0.50 [0.21] hours vs 6.83 [3.19]ours) and had a mean Cmax approximately 10-foldigher than the commercial 5-mg cyanocobalamin for-ulation (12,847 [6613] pg/mL vs 1239 [450] pg/mL).limination of B12 occurred at approximately theame rate for both formulations. Mean Ke was 0.025
[0.009] and 0.028 [0.006], whereas t1/2 was 30.06[8.24] hours vs 25.95 [6.07] hours for treatments B andC, respectively. As noted in Table III, it was possible toestimate the elimination phase in 5 out of 6 subjects fortreatment B and in 3 out of 6 subjects in treatment C.
Estimates of %F were performed for all oral treat-
ments (A, B, and C) and are shown in Table IV. AllVolume 33 Number 7
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values are reported as mean (SD). Estimates of %Fwere also performed using baseline-adjusted B12 con-centrations and with or without body weight and BMIadjustments (Table IV).
Statistical comparison of B12 %F with and withoutaseline adjustment and either with or without bodyeight adjustment in Table IV indicates that 5-mg cy-
anocobalamin/SNAC (treatment B) had significantlygreater %F compared with treatment C (P � 0.005).B12 %F was slightly less following baseline adjustmentcompared with the non–baseline-adjusted data (4.24[1.28]% vs 1.40 [0.66]%, for baseline-adjusted treat-ments B and C, respectively; 5.09 [1.53]% vs 2.16[0.78]%, for baseline-unadjusted treatments B and C,respectively). In treatment C, however, B12 non–base-line-adjusted %F was significantly greater than base-
Table II. Participant demographic characteristics and
Parameter A
Gender, no.Male 4
Race, no.Black 4Caucasian 0European/Middle Eastern 0Hispanic 0
Age, yMean (SD) 33 (7)Range 28–43
Weight, kgMean (SD) 69.5 (5.3)Range 65.0–77.0
Height, cmMean (SD) 170 (6)Range 163–175
BMI, kg/m2
Mean (SD) 24.0 (1.8) 2Range 21.8–25.8
Cyanocobalamin baseline, pg/mLMean (SD) 534 (247)Range 341–878
BMI � body mass index.
line-adjusted %F after values were normalized for BMI
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(2.15% vs 1.27%; P � 0.05). Generally, %F was notaffected by body weight or BMI adjustment.
TolerabilityThere were no AEs reported in this study with any
treatment. Mean clinical laboratory values, vital signs,and ECG parameters remained within normal limits atthe end of study, with minimal changes from baselineand no important treatment-related differences ob-served. No changes in physical examination resultswere reported. All treatments were well tolerated bythe healthy male participants, with no apparent differ-ence in safety profile by treatment. Administration ofall study treatments, including the single oral doses ofcyanocobalamin/SNAC up to 10 mg/200 mg (treat-ment A), appeared to be well tolerated by the
line cyanocobalamin concentrations.
Treatment
OverallC D
6 4 20
5 3 151 1 30 0 10 0 1
8) 33 (9) 24 (4) 30 (8)43 23–45 20–30 20–45
11.0) 74.1 (8.5) 78.5 (13.5) 76.7 (10.5)94.4 61.3–85.0 66.0–93.3 61.3–94.4
4) 172 (5) 176 (3) 174 (5)180 165–178 173–180 163–180
3.0) 25.1 (3.2) 25.2 (3.3) 25.4 (2.9)30.0 22.3–29.8 22.1–28.7 21.8–30.0
193) 387 (118) 666 (294) 512 (216)837 218–474 408–1084 218–1084
base
B
6
3111
29 (22–
83 (67.1–
176 (170–
6.7 (23.0–
518 (267–
participants.
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Clinical Therapeutics
DISCUSSIONA tablet formulation of cyanocobalamin containingSNAC, an absorption enhancer, (cyanocobalamin/
Table III. Pharmacokinetic parameters of non–baseliA, B, C, and D.
PharmacokineticParameters
Treatment A* Tre
Mean (SD) n Mea
Cmax, pg/mL 28,175 (13,681) 4 12,847Tmax, h 0.54 (0.32) 4 0.50AUC0–last, pg/mL/h 127,494 (65,790) 4 54,609AUC0–�, pg/mL/h �
Ke, 1/h 0.03 (0.010) 3 0.025t1/2, h 25.31 (8.8) 3 30.06
SNAC � sodium N-[8-(2-hydroxybenzoyl)amino]caprylate.*Treatment A: 2 � 5-mg cyanocobalamin/100-mg SNAC ta†Treatment B: 1 � 5-mg cyanocobalamin/100-mg SNAC ta‡Treatment C: 1 � 5-mg cyanocobalamin tablet.§Treatment D: 1-mg cyanocobalamin IV (1 mg/mL solution�Values missing or not reportable.
Figure. Time course of mean (SD) serum concentratiosubjects (logarithmic scale).
942
SNAC), was compared with a marketed immediate-release tablet containing the same quantity of cyano-cobalamin (5 mg) in a small single-dose study in
rrected serum cyanocobalamin following treatments
t B† Treatment C‡ Treatment D§
) n Mean (SD) n Mean (SD) n
3) 6 1239 (450) 6 221,287 (80,248) 4) 6 6.83 (3.19) 6 0.05 (0.03) 405) 6 23,165 (8382) 6 214,738 (44,614) 4
� 235,165 (43,854) 39) 5 0.028 (0.006) 3 0.048 (0.018) 3) 5 25.95 (6.07) 3 15.53 (4.70) 3
treatments B, C, and D administered to healthy male
ne-co
atmen
n (SD
(661(0.21(16,4
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(0.00(8.24
blets.blet.
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Volume 33 Number 7
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M. Cristina Castelli et al.
normal healthy male subjects. This study found thatSNAC enhanced the absorption of cyanocobalamin.The cyanocobalamin/SNAC formulation also resultedin a shorter Tmax compared with the commercial cya-nocobalamin formulation. The group mean Tmax was.5 hours for cyanocobalamin/SNAC and 6.8 hoursor the commercial cyanocobalamin formulation. Inddition, cyanocobalamin/SNAC mean peak B12 con-entrations were �10-fold higher (12,847 pg/mL vs239 pg/mL). The calculated t1/2 of B12 was similar
with both formulations. Absolute bioavailability ofB12 with the SNAC formulation was calculated fromnon–baseline-adjusted data to be 5.09% and was sig-nificantly greater (P � 0.05) than the commercial for-mulation (2.16%). There were no tolerability-relatedfindings in the study.
The study population consisted of 75% black malesand no women. Studies of ethnic differences in use ofcobalamin and its metabolism conducted in the UnitedStates21,22 have indicated that blacks have significantlyhigher endogenous cobalamin levels than Caucasians,and a trend toward higher endogenous cobalaminlevels in female subjects has also been reported. Thedesign of the present study did not evaluate the im-
Table IV. Treatment B versus treatment C per-cent bioavailability (%F) non baseline-adjustment.
ParameterTreatment B
(n � 6)Treatment C
(n � 6)
%F (AUClast) 5.09 2.16T Test value 4.18P-value 0.0019%F (AUClast_D) 5.5 2.02T Test value 4.36P-value 0.0014%F (AUClast_BMI) 4.65 2.15T Test value 4.02P-value 0.0044
%F (AUClast)- Percent bioavailability without body weightadjustment.%F (AUClast_D)- Percent bioavailability with body weightadjustment.%F (AUClast-BMI)- Percent bioavailability with BMI adjust-ment.
pact of gender and ethnicity of the subjects. How-
July 2011
ever, the PK response to both treatments in the pres-ent study exceeded baseline values in all cases for theduration of the study and clearly demonstrated dif-ferences in bioavailability. Individual B12 predosevalues ranged from 218 to 1084 pg/mL in this smalland selected healthy subject study population. Thechoice to test only 1 gender was made to limit vari-ability, given the small sample size.
The oral cyanocobalamin dose of 5 mg used in thecontrol group (treatment C) resulted in B12 %F ofslightly more than 2% in the present healthy male andpredominantly black population. To our knowledge,this is the first study reporting %F of oral formula-tions. A study conducted in 1968 reported 1.2% bio-availability in a wide range of cyanocobalamin doses(100 �g–100 mg) in a Swedish population.3 In thisaper the method used to calculate bioavailability wasased on the relationship between dose and urinaryxcretion of radioactive B12.
SNAC has reportedly low toxicity for animals. In a3-week general toxicity study in rats, a no-adverse-ffect level of 1000 mg/kg has been reported.9
The specific mechanism of action of SNAC en-hancement of cyanocobalamin absorption has notbeen studied. However, studies with several differentpolar drug molecules have shown a single pattern ofSNAC action, which may also be relevant to cyano-cobalamin. Laboratory studies of insulin,23 hepa-in,24 cromolyn,25 and human growth hormone26
when coformulated with SNAC have shown that thecarrier aids gastrointestinal absorption by fluidiza-tion of gastrointestinal epithelial cell membranesand transcellular uptake of both drug and carrier.Membrane fluidization by SNAC is concentrationdependent and reversible.
Clinical studies of SNAC administered with heparinresulted in enhanced heparin absorption and rapid on-set of action as measured by pharmacodynamic re-sponse (activated factor Xa).11 The Tmax of both hep-arin and SNAC was measured in healthy male subjectsfollowing a single dose of SNAC/heparin.27 The Tmax
values for SNAC and heparin were similar at 0.50hours and 0.58 hours, respectively.
Both oral and intramuscular routes of administra-tion of B12 supplementation are in clinical use. Inmost countries, including the United States, intra-muscular treatment is the classical treatment for B12deficiency.2 Oral treatment is more convenient and
less expensive to administer, but questions remain943
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Clinical Therapeutics
about its reliability.28 Based on a search of MED-INE, there appear to be no other reports of effectivenhancement of oral B12 delivery with permeationnhancers.
Further controlled clinical studies in B12-deficientatients of cyanocobalamin/SNAC and current B12 re-lacement regimens are needed to establish the effec-iveness of this new approach to normalizing serum12 and the major biomarkers of B12 deficiency, meth-lmalonic acid and homocysteine.2 The present studyas designed with a small group size of 4 to 6 mainlylack males and, therefore, confirmatory studieshould be conducted with a larger and more diverseopulation.
A single measurement of baseline serum vitamin12 was taken from the members of the study popula-ion. In similar studies involving the PK analysis ofndogenous substances, several measurements of base-ine values are recommended to improve accuracy.17
However, a single measurement is adequate whendoses are high and the concentrations produced clearlyexceed baseline, as in the present study.
Crystalline B12 has an oral bioavailability of �1%ver a wide dose range.3 Dose proportionality of the
test formulation has not been established. However, inthe present study, doses of 5 and 10 mg resulted in anapproximate doubling of AUC and Cmax (Table III).
Changes in the PK profile of crystalline B12 whenosed in the test formulation consisted of an earlier
max and greater bioavailability compared with theonventional oral formulation. Similar PK profilesave been observed with other drug formulations usinghe same technology.23–27 However, studies to deter-
mine the changes, if any, to B12 site of absorption anduptake mechanism, both potentially important consid-erations for a new B12 oral therapy, have not beenperformed.
CONCLUSIONSAn oral formulation of cyanocobalamin (5 mg) con-taining SNAC (100 mg), an absorption enhancer, pro-vided significantly improved bioavailability and a sig-nificant decrease in time to Tmax in a small study oformal, healthy male subjects compared with a com-ercially available oral formulation (5 mg cyanoco-alamin). Both oral formulations and an IV cyanoco-alamin formulation (administered at a 1-mg dose),
ere well tolerated.944
ACKNOWLEDGMENTSThis study was financed by Emisphere Technologies,Inc, which also manufactured and supplied the SNACtest formulation. The authors are or were employees ofEmisphere Technologies. Drs. Castelli and Riley arecurrently employed by Emisphere Technologies andmay benefit from the success of products arising fromthis work. The authors have indicated that they haveno other conflicts of interest regarding the content ofthis article.
Carol Thomas-Sharp monitored the study; KarenBrazzillo and Kiran Chaudhary were responsible forproject management. Moses Oyewumi, Matt Gurler(deceased), Jun Liao, and Prateek Bhargava contrib-uted with clinical trial dose preparation. James Sherry,MD, PhD, was product safety physician. Joe Fotso re-viewed the validation report for B12 analysis fromMDS Pharma Services. MDS Pharma Services con-ducted this clinical study, performed the bioanalyticaldeterminations, and wrote the study report.
Dr. Castelli and Ms. Wong designed the study, re-viewed the pharmacokinetics and safety analysis, con-tributed to the interpretation of data, and drafted themanuscript. Ms. Friedman monitored the study. Dr.Riley made substantial conceptual contributions andrevisions.
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Address correspondence to: M. Cristina Castelli, PhD, Emisphere Technol-ogies, Inc, 240 Cedar Knolls Road, Cedar Knolls, NJ 07927. E-mail:
945