AD-P008 842 94-08720

ORAL PHARMACODYNAMIC BIOAVAILABILITY OF SiXp-AMINOPEENONZ DERIVATIVZS

Larry D. Brown, Thomas G. Brewer, Douglas R.Flanagan*, Jurgen D. von Bredow, Ralf P. Brueckner,

and Dr. Robert R. Engle.

Division of Experimental Therapeutics, Walter ReedArmy Institute of Research (WRAIR), Washington, DC

20307-5100;*School of Pharmacy, University of Iowa, Iowa City,

Iowa 52242.

ABSTRACT

A number of potential biochemical approaches existfor protection against and treatment of cyanidepoisoning. The methemoglobin protective approachappears to be the most promising and practical.Adequate protection against the lethal effects ofcyanide intoxication depends upon a sufficient amountof available methemoglobin to bind and remove the toxiccyanide ion from the circulation. The intravenousadministration of solutions of direct and indirectmethemoglobin formers WR000302 (p-aminopropiophenone,PAPP), WR269,410 (p-aminoheptanophenone, PAHP),WR258,948 (p-aminooctanophenone, PAOP) and theirrespective N-hydroxy derivatives (WR270,101, WR272,677and WR271,159) to unanesthetized beagle dogs leads to arapid increase in whole blood methemoglobin. In theabsence of a specific analytical method for theaccurate analysis of each of these six compoitrds, thecorre3ponding whole blood methemogPebin induced by eachof these compounds were monitored by the RadiometerOSM-3 hemoximeter immediately following blood samplingto construct a pharmacodynamic profile of methemoglobinvs time. When these same animals were given oralgavage doses of either a solution or suspension of thesame propiophenone derivative, a rapid and consistentincrease in methemoglobin levels was also observedwhich may be used as a measure of the rate and extentof absorption of the compound into the whole blood

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circulation. The pharmacodynamic profiles of theintravenous dose can be compared with the oral gavagedose of the two formulations (solution and suspension)to obtain an estimate of pharmacodynamic oralbioavailability. This estimate can be used to selectthe compound and formulation which provides thenecessary duration of methemoglobin sufficient toprotect against possible exposure to cyanide.

INTRODUCTION:

HCN is a chemical warfare threat agent. Cyanide (CN-)is an intracellular poison that binds to cytochromeoxidase, thereby disrupting the electron transportsystem. This interruption of cellular respirationblocks ATP production and rapidly leads to cell death.Hydrogen cyanide (HCN) is highly volatile and wheninhaled by man in high concentrations causes dizzinessand confusion within seconds. Respiratory arrest mayoccur in less than one minute. MOP? gear providessoldier protection if donned immediately and gas maskfilters remain functional.

Current cyanide treatment requires expedientintravenous administration of approved drugs (sodiumnitrite and sodium thiosulfate) which, on thebattlefield, is impractical. Therefore, prevention ofcyanide-induced casualties by prophylactic measures isessential. Drugs that induce methemoglobinemia havebeen recognized as effective cyanide antidotes foryears. Methemoglobin (MHb) contains an oxidized(Fe3+) form of heme iron that has a higher CN- bindingaffinity than does cytochrome oxidase. Preferentialbinding of CN- to MHb to form cyanomethemoglobinremoves it from the cytochrome oxidase active site andrestores normal cell respiration.

A safe, well tolerated, and effective prophylactic forsoldiers who may be exposed to cyanide is needed. Theideal treatment should provide adequate protection(against at least 2 LD505 of cyanide), be easilyadministered (preferably orally) at daily or lessfrequent intervals, and not interfere with soldiers'performance.

Methemoglobinpmia is known to protect mammals againstlethal doses of cyanide. It is necessary to evaluatethe kinetics of methemoglobinemia induced by candidatedrugs to determine whether methemoglobin formation andelimination rates are rapid enough as well as

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predictable and stable enough to be used in a longterm dosing regimen in soldiers. Bioavailability isone of the most important ,arameters for drugdevelopment candidate advancement decision making.Formulation modifications can enhance thebioavailability of orally delivered drugs but gains inthis area are limited, therefore candidates with thebest inherent absorption must be identified.Bioavailability is defined by the FDA as both theextent (relative amount) of drug from an administereddosage form which enters the systemic circulation andthe rate at which the drug appears in the blood streamand presumably at the site of dynamic action. Threety'•s of bioavailability (absolute, relative, relativeý.rcimal bioavailability) are described in the'.iterature. Absolute bioavailability (F) is calculatedLy the following formula: F = AUCpo/AUCiv and is thefraction of drug absorbed by any nonvascular route inrelation to the intravenous dose which is assumed tobe 100%. Absolute bioavailability have values whichrange from 0 < F < 1. A drug with an F value of 1 has'perfect' or 100% oral bioavailability equivalent todirect intravenous infusion.

Six p-aminophenones were studied in the male beagledog to determine the single dose time-concentrationcurves for methemoglobin formation after intravenousand stomach gavage (POG) routes. Bioavailab.ility (F)was calculated for both solution and suspensionformulations of these six compounds to -allowadvancement rank ordering based upon t), s data inconcert with other available informat.'uoi and forimprovements in formulation, if needed, for future

* planned pharmacology studies.

SThis study evaluated the utility of using 'effectkinetics' or the pharmacokinetics (PK) of a dynamiceffect, i.e., methemoglobin (MHb) formation as an

* estimate for drug or metabolite bioavailability and as!" a surrogate for drug time-concentration data.

, MATERIAL3 & METHODS:

!, Single intravenous and oral gavage formulations of sixcandidate p-aminophenones were administered to beagledogs. This effort required intensive blood samplingand time-sensitive analysis of large numbers ofsamples. We routinely conducted a study using 4animals each day with immediate measurement of MHbvalues. This manuscript is based upon data collectedfrom 72 dog experiments and approximately 1,450 blood

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samples. Information was collected from each animalduring three separate experiments (intravenous, gavagesolution, gavage suspension) using a crossover design.

CHEMICAL COMPOUNDS:

PAPP, PAOP, PAHP (indirect methemoglobin formers) andthe respective N-OHs (direct formers) for these threeaminophenones were obtained from GMP certifiedinventory stock which had been synthesized under Armycontract for the WRAIR Experimental TherapeuticsRepository (over 275K distinct chemical compounds).

0 0

M.W. 140.13. C,, 1 0 MW. * 162.Z C1,411 "

P-A W10WHAyOW"ImePAHP, Vl2Ig10U.W. .206.3, CJIAO" ktW. 221.3 C.,k4,,M

M.W,. *211.3. C,,142,NO K.w. - M231 C,ý$,1NO

SELECTION and CARE OF MODEL:

The dog model is the most similar to humans and themost predictive of methemoglobin forming as well asclearing activity in humans. Rodents are poor MHbformers. Methemoglobin reductase activity issubstantially higher in mice than in humans. TheBeagle dog has been used in previous pharmacokinetic/pharmacodynamic studies of methemoglobin withexcellent success and all available data to this pointfor MHb formation with 8-aminoquinolines derives fromthe dog model. Ruminants form methemoglobin fasterand more extensively than other large mammals although

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rthe MHb-reductase activity of adult dogs and sheep issimilar. The presence of the rumen substantiallyalters the absorptive physiology of sheep so theycannot be used as a model for orally dosed drugs.Swine have dissimilar methemoglobin reductase levelsto those in humans.

A total of 28 domestic born purpose bred young adultmale HRA Strain Beagle dogs (Canis familirls)obtained from Hazelton Research Products weighingbetween 7 and 14 kq were used in these studies.Animals were one-two years of age, identified by eartatoo, and housed at the WRAIR. Animals were caredfor under veterinary supervision in accordance withthe principles in the guida for tho Care and U..ofLaboratorv Animals (Department of Health, Educationand Welfare Publication, NIH 86-23).

DOSING PROCEDURES:

Oral and intravenous dosing formulations were preparedfresh in either a PEG 200 solution or 2%carboxymethylcellulose (CMC) suspension. For oralgavage dosing, a Surgilube® coated 16 French Salemsump gavage tube was passed per os into the stomach inthe consious animal. The dose was administeredfollowed by a flush with 10 ml of tapwater. Dogs wereobserved closely fcr 4 hours after each dose foremesis. Vomiting was not observed in the 46 gavageddogs. Dogs were held NPO (for food only) for 12 hoursbefore each dose and 4 hours afterwards (food andwater). For intravenous dosing, the drug was preparedin filter sterilized polyethylene glycol (PEG) 200(Sigma Chemical Co). Depending upon the solubility ofeach drug and the animal weight, intravenous volumesof 5 to 12 mill 4 liters of PEG solution containing drugwere infused over a three minute period into anindwelling in'ravenous catheter using a Harvard Pump,followed by a flush of 10 milliliters of heparinizedsaline. Dose levels studied included: 0.4 mg/Kg forPAPP, 6.0 mt Kg for PAHP, 15.0 mg/Kg for PAOP andisomolar doscs (0.75,1.0 and 1.06 mg/Kg, respectively)for the N-hydroxy derivatives or putative metabolitesof these three parent phenones.

BLOOD SAMPLING and ANALYTICAL METRODS:

Samples were normally collected from the contralateralcatheter to the one used for infusion. Venous wholeblood samples were collected in neparinized syringes.0.5 ml of 'waste' blood was removed from the catheter

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prior to collection of the measured sample to removeheparinized saline flush solution diluted blood.

Samples were immediately measured for total hemoglobin(gms %), HbO2 (%), HbCO (%), MHb (%) and O2ct (vol%)using the OSM3 Co-Oximeter (Radiometer America,Copenhagen) and then placed on wet ice until theycould be spun down for plasma collection and freezing(generally within 30 mins) . Whole blood and plasmasamples for drug concentration determinations werefrozen at -20 0 C and stored for future analysis whenvalidated methods become available.

DATA and STATISTICAL ANALYSIS:

Methemoglobin time-concentration area-under-the-curve(AUC) determinations were made using the 'trapezoidalrule.' Non-linear non-compartmental 'least squares'regression analysis was performed on MHb data fromthree separate experiments (IV, gavage solution,gavage suspension) on each dog using the MicrosoftEXCELT- LINEST function to obtain AUCs, %AUC, MHbmax,Tmax, disappearance rate, disappearance constant, andstandard statistics (Sey, F, df, SSreg, SSresid,slope, y-intercept). The oral gavage AUCo-- at agiven 0:.e of each drug was divided by the intravenousAUCo-o at the same dose (mg/Kg) for each animal tocalculate MHb bioavailability (F).

RESULTS:

Data from three separate experiments on dog DFWABdosed with PAHP-OH are graphically represented asthree methemoglobin time-concentration curves inriqure 1. AUCo-- data from these three experimentswere entered into the appropriate formulas (Fl -AUCpogsoin/AUCiv; F2 = AUCpogsusp/AUCiv) forgenerating two separate bioavailability estimates fordrug in each animal.

PI P , I , .,

. 8 . 4

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Summary data for 26 dog experiments (20 individualanimals; 6 were used twice) which received intravenousand gavage solutions and 20 dogs which also receivedgavage suspensions are offered below in Table 1.Group mean bioavailability values (Fl for solution; F2for suspension) for PAPP, PAOP, PAHP-OH, PAOP-OH,PAPP-OH (N=4) and PAMP (N=6), sample standarddeviations and percent coefficient of variation (CVgroup SD/group mean) were calculated.

TABLZ 1. Group Mean~ Rioavailability (r) ±sD Ccv)

C~ndid.IDm r ~ Md B.g. oo l A !pg MAUCI,* FZ. AUCpo~gsu.pwAUC.j

PAIP I M~tf [n.6) ________

-MIanwmak...SO)gn-y) L 0442 t 0 1 4 3 4..- 0.393 0 015138

(3 ~ ~ U t4b~ M~o SO LCýj 06209 0 311L 581 3L5904 3 722)

PAJIP. I5 mK (n.4)

(8cto ~qW- Dý 047 - - ____

[N-ýdro PAýe) U..o-LýL 1.. 0 OLDSL 0.041029)

__mev ____ _Sl).S.p!pS AU~p~pqn.AUý vag PEG 2oq00 wo9

The six phenone drug candidates were then rank orderedbased upon oral bioavailability tor solution (Fl) andsuspension (F2) formulations (Table 2).

TARLIZ 2. Phenon. Ran~k Ord~r

Candfdate FRank Order _SoM Absolute Bioavailff'_) SuSp Abzoulut Imoqvvait (F2) SuspBioavei( Horno eneily

1 Best PAHP-OH PAHP PAPP2 PA)PPOH PAPP PAOP.0143 PAPP PAOP-Oti PAPP.OH

4PAHP PAHP-OH PAHP

PAPO

6 875ed PO NtCndce

gl!" Mft oilnto ewn V

DISCUSSION:

As expected, intravenous dosing resulted in thehighest MHb levels (peak or Tmax) and the largest AUCs(F = 1.0), followed by gavage solutions andsuspensions in that order (Figure 2). Gavagesuspensions of PAOP were not attempted due to the verylow levels of MHb observed with the PAOP gavagesolution.

SI j , iilli i llii iJaww I i-I i.l IOQI I. wo

Consistent oral bioavailability is an importantconsideration in development of any pharmaceutical andwas best for PAPP (Figure 3). The suspension is aspecial case of the aqueous solution which providesdata on whether the drug can be delivered in tabletform. Based upon the evident absorption of PAPP andPAHP from these gavage suspension trials, an effectivetablet formulation of these candidates would bepossible.

h.... I3,

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Rank ordering of candidates depended heavily upon thetype of formiulation. For instance, the 'leadcandidate' (PAHP-OH) from the solution experimentschanged to PAHP after the suspension experiments(Table 2). Additional information for final drugcandidate selection will depend upon the stability ofeach of the compounds and the aqueous solubility overa range of pHs associated with the gastrointestinaltract (e.g., pH-water solubility profile).

These data may be used to support recommendations forrational selection of a suitable candidate drug forfurther development as an anti-cyanide prophylactictreatment. In addition to this bioavailability data,candidate advancement or transition and selection willdepend upon other factors such as physiochemistry(stability, solubility), genetic and general mammaliantoxicology outcomes, success of analytical chemistrymethod development, metabolism (ADME), PK/PD [durationof drug levels or dynamic effect (tl/2s), potency(Tmax), clearance (CL), Vd, Ke, AUC], dose-responsedata, and additional formulation considerations.

CONCLUSIONS:

We have applied 'effect kinetics' or pharmacodynamicprofiles of drug effects to estimate drugbioavailability and aid initial selection of c3ndidatedrugs in the devel)pment process. A precedent was setfor this approach in prior Army research onpyridostigmine, a prophylactic pre-exposure drug forprotection against nerve agent poisoning. Kinetics ofthat effect, i.e., reversable anticholinesterase(AcHE) enzyme inhibition, was used to rank ordercandidates in that project in lieu of drug levelkinetics.

• Based upon the use of this concept, we have rankordered six candidate cyanide pretreatment compounds.Drug pharmacokinetics will eventually have to bedetermined but only on the 'lead compounds' and notfor all six candidates.

• On an absolute bioavailability basis, the N-hydroxyderivatives of PAP? and PAHP are the best candidates,however, from the practical requirement for deliveryby oral tablet formulation the parents of these twocompounds (i.e., PAPP and PAHP) will be the superioralternatives.

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RzFZRZNCES:

* Ritschel WA. Handbook of Basic Pharmacokinetics.Hamilton, IL: Drug InteIligence Publications, Inc.,1986.

* Rowland M, Tozer TN. Clinical Tharmacokinetics:Concepts and ADolications. Philadelphia, PA: Lea &Febiger, 1989.

* Ford B. Methemoglobin inducers Ir: Calabrese, E.J.Princioles of Animal Extrapolation. New York: JohnWiley and Sons, 1983.

S• Gibaldi M, Perrier D. Pharmacokiaetics. New York:*. Marcel Dekker, Inc., 1982.

* Ballantyne B, Marrs TC (Eds). Clinical andExporimental Toxicology of Cyanides. Bristol, England:Wright Publishing, 1987.

• Kiese M. Methemoglobin. Boca Raton, FL: CRC Press,1974.

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