Burckart, GJ

Makowka, L

Sanghvi, AT

Selftman, H

Starzl, TE

Todo, S

Venkataramanan, R

Warty, VS

Zemaitis, MA

English

D-Scholarship@Pitt

Biopharmaceuticai Aspects of FK-506 R. Venkataramanan. V.S. Wa .. ~DEK M.A. Zemaitis. A.T. Sanghvi. G.J. Burcltart. H. Seltman. S. Todo. L. Makowks. and T.E. Starzl FK-506 (FK) is a novel immunosuppres-sant isolated from Streptomyces tsuku-baens;s (Fig 1). It is nearly 500 times more potent than cyclosporine (CyA) in inhibiting lymphocyte proliferation in mixed lympho-cyte cultures. I In vivo. it significantly prolongs the graft survival in rats receiving heterotopic heart transplantsl and dogs receiving renal l or liver transplants.4 PHYSICOCHEMICAl PROPERTIES FK has a unique chemical structure with a molecular weight of 822.05. FK is fairly lipid soluble and is similar to CyA in its solubility properties. It is poorly soluble in water and hexane but very soluble in methanol. chloro-form. ethyl acetate. ether. and acetone. In the solid state FK is stable for at least 6 months at 240C and 40°C. A methanolic solution of the drug appears to be stable for at least 4 weeks at - 20°C. for 2 weeks at 5°C. and about I week at 24°C. A methanolic solution of the drug decomposes into multiple components in presence of 6 N HCI. However. FK is stable in the presence of very dilute acid. DOSAGE FORM Several dosage forms of FK were used in our studies. The intramuscular dosage form contains 50 mg of the drug, 10 mg of the surfactant HCD·60. and 125 mg of mannitol. This preparation is used after the addition of a required amount of normal saline and sonica-tion for 15 minutes at room temperature. The oral formulation is a solid dispersion of the From ,h, Schools 0/ Pha,maC'" aNi M,dici",. Uniwr-sit,.. 0/ Pltubu"h. Add',11 rtp"'" "qUtSIS'O R. V,,,lcotllramlllUJ". PhD. U,,;wrllly of Plttsbur,h Ciinicill PllarmaC'olci","cs LAb-ora,,,,,... 807 Siliit. Hilli. Pittsbur,h. PA 1.5261. e 1987 hy aru", ci Stratlon. IIIC'. 004/-1 J45/87/1905.fJ()()6S0J.OO/O drug (20%) that is either suspended in water for studies in rats or filled in a soft gelatin capsule for studies in dogs. For intravenous studies. the intramuscular dosage form is diluted in a mixture of alcohol, Cremophor EL (BASF Wyandotte Corp. Parsippany, NJ). and normal saline. DRUG ANAlYSIS FK does not contain any chromophores. A methanolic solution of the drug absorbs UV light to the maximum extent (A",..) at 192 nm. The compound also does not volatilize readily and has no intrinsic fluorescence. Therefore. traditional analytic methods such as gas chro-matography or gas chromatography-mass spectrometry cannot readily be used to ana. Iyze intact FK in biologic fluids. FK can be measured by high-pressure liquid chromatog. raphy (HPlC) or by an enzyme immunoassay (EIA). HPLC Analysis HPlC assay can be used for FK measure· ment under certain circumstances. Because a number of components absorb UV light at 19i nm. a wave length of 210 or 214 nm has to be used for measuring FK by HPlC. This results in a low sensitivity for measuring the drug in biologic fluids. The HPlC assay is linear over a concentration range of 50 to 1.000 ng/mL and requires at least 1 mL of the biologic fluid. This assay can be used to test the stability of the dosage form of the drug. £nzym~ Immunoassa.v I n this assay. the drug is extracted from biologic fluids with benzene after the addition of 0.2 mol/L phosphate buffered saline (PBS. pH 7.0). The benzene extract is reconstituted in a mixture of 1:4 peroxidase-FK conjugate (FK·POD) and 0.05% Tween 20 and 0.5% 30 qIKKKKKI~~ 1/01 XIX. No 6. Suppi 81Octot.l. 1987: lIP 30.36 BIa"HARMACEUTlCAl ASPECTS OF FK-506 • H,O Fig 1. Chemicalltructura of FK-&08. bovine serum albumin in PBS (T-BSA-PBS). Before this. 200 ~i of the polyclonal FK-antibody solution in PBS is added to each well of a 96-well fiat tissue culture plate and incubated at 4°C overnight. The plates are then washed multiple times with 0.05% Tween 20 in PBS. Approximately 300 ~i of T-BSA-PBS is added to all the wells and incubated at room temperature for two hours to suppress any nonspecific binding of FK-POD to the plastic wells. The plates are washed with 0.05% Tween in PBS and filled with PBS. This solution is aspirated. the reconstituted extract is added to each well. and the plates are incubated on a plate rotor overnight at 4°C. The solution in the plates is aspirated. the substrate solution (1.0 mg/mL of O-phenylenediamine dihydrochloride and 0.015% H101 in phosphate-citrate buffer. pH 5.4) is added. and the plates are incubated at room temperature for 20 minutes in the dark. Fifty microliters of 4 N H,80. is added to stop the reaction of the peroxidase enzyme with H20} and O-phenylenediamine. The optical density is measured at 492 nm. The concentration of FK in the sample is calcu-lated from a standard curve developed by using known amounts of FK. The mmimum detectable concentration of FK when using EIA is 0.1 ng/mL. A standard curve is constructed and used over a concen-1.6 14 1.2 >-!:: II) z ~ 1.0 ..... c u i= .8 0.. o .6 .4 .2 31 o I 10 100 1000 FK-506 CONCENTRATION (NG/MLI Fit 2. Stllndilrd curva of pIa_ fK concentration v optical daMity at 492 nm. tration range of 0.1 to 100 ng/mL (Fig 2). Samples with a concentration of greater than 100 ng/mL are diluted in PBS and then analyzed. PHARMACOKINETICS Absorption Oral administration. Oral absorption studies of FK were carried out in male beagle dogs eight days after daily oral administration' of FK in a soft gelatin capsule. Blood samples were obtained from the femoral vein at O. I. 2. 3. 4. 6. 8. 10. 12. 16. and 24 hours after drug administration. Figure 3 shows the plasma concentration-olf-time profile in a dog after oral administration of 1 mg/kg of FK. After oral administration of I. 2. or 4 mg/kg of FK. the trough plasma concentrations were 0.48. 1.4. and 6.8 ng/mL. respectively. There was a wide variation in the trough plasma concen-trations of FK in animals receiving the same oral dose.<0.25 to 0.8 ng/mL after a I-mg/kg dose and 0.2 to 2.3 ng/mL after a 2-mg/kg dose). The drug was rapidly absorbed. with ._-----_ .. _----32 2 ~ 12 z 9 ... .. II: .. ~ 08 ~ o u .. ~ 04 .. -' Q. o~~~--~~--~~~~~~~ 2 4 6 8 10 12 14 16 18 20 T111. ,HOURS, Fit 3. PI .. me --.metlon of FK In. cloG (no. 2) on dIIy 8 efter d.iIy or.,.dm/nl.,r.tion of' me'kg of FK. the time for maximum blood concentration (t...) of one to three hours. This indicates that the drug is absorbed from the proximal part of the small intestine. However. after 4 mg/kg. there was prolonged absorption of the drug, and peak plasma concentrations were ob-served only six to ten hours after drug admin-istration. The specific reasons for this observa-tion are not clear at this point but may be related to potential gastric motility changes mediated by the drug. The maximal plasma concentrations (emu) ranged from 0.8 to 1.8 ng/mL after a I·mg/kg dose and from 3.2 to ~ I § 12 I .. cr a: .. z w ~ 08 o u cr 2 rJ'I ~ 04 0.. VENKATARAMANAH ET Al. 5.3 ng/mL after a 2·mg/kg dose. When absorption studies were conducted on day 15 after daily FK administration. a general ten-dency for emu and T mu to increase was observed. which indicated changes in the rate and the extent of FK absorption with time. In some dogs, secondary peaks in plasma concentrations were observed (Fig 4). The specific rcason(s) for this phenomenon is not clear at this time. In theory, this may he due to enterohepatic recirculation of the drug or biliary excretion of a conjugated metabolite of the drug that is hydrolyzed in the gut and is reabsorbed. Precipitation of the drug due to tbe local environment. such as pH changes, and subsequent reabsorption can also be responsible for sucb an observation. Differen-tial absorption from different segments of the gut could also contribute to secondary peaks. Studies in animals where bile can be diverted from the gut.l and further studies of the pH-solubility profile of the drug are needed to I characterize this process. The area under the plasma concentration-· v-time curve (AUe) as measured on day 8 after oral administration of 1. 2. or 4 mg/kg of FK is shown in Fig 5. This figure indicates a disproportionate increase in AUe with a 2 4 6 8 10 12 14 16 18 20 22 24 Fig 4. PI ...... _ .. lion o. FK In • dOO (no. JI on My 8 .fter d •• 1y or.,.dmiI_a ....... of' mG/kG of Fl(. TIME (HOURS) BIOPKARMACEUTICAL ASPECTS OF FlC-506 2'Or 2°Ol ..... 160 ... 1 ~ I ga 120 ~ ..... ~ u ~ ~UM 40 l~--~~--~O~--~P~--~4 DOSE (mg/kg) Fig Ii. Ar .. und ... the ple_ FK COnaentnition-lI'-drne curve eft ... or .. aclnrininratlon of ,. 2. and 4 ""III, of FK. small increase in the dose. This may be due to saturation of the drug-metabolizing enzymes responsible for FK elimination. saturation of the tissue uptake of the drug, or prolonged or sustained presence of the drug at the site of ..J ~ ..... t!) ! z o ~ 12 a: I-z W u z 8 08 « ~ (/) « ~ 8 04 ~ '" ... 33 absorption. thereby increasing the fraction of the dose that is absorbed after a 4-mg/kg dose. Intramuscular administration. FK was administered intramuscularly (0.49 mg/kg) into the thigh muscle of two male beagle dogs. Blood samples were obtained at O. I. 2. 3. 4. 6 • 8. 12. 16. and 24 hours after drug administra-tion. FK was slowly absorbed from the injec. tion site. as indicated by a slow and continuous increase in the plasma concentration of the drug over the entire study period in both dogs (Fig 6). Distribution Preliminary studies indicate that FK is distributed into RBCs. FK. however. does not alter the uptake of CyA by human RBCs. This is indicated by a blood-ta-plasma ratio of 1.5 for Cy A in the presence of 500 ngi mL of FK and in the absence of FK. Studies are currently under way to examine the binding of FK to various components of blood and to tissucs. Elimination Studies have been carried out in rats and dogs after various routes of administration to determine the urinary excretion of FK. Inde· pendent of the route of administration. less than I % of the dose of the drug appears in the .---A--________ .'OG. " '------- '.lo OOG'11 Fig e. PI .. ",a COMMItra-lion of FK In dot no. 10 161 and dot no. 11 101 aft... a .. .,... In_·' ..... int-iOn of 0.41 "'9/11,ofFK. l£-~~~~~--~~~~±_~~=_~~ 2 4 6 8 10 12 14 16 18 20 22 24 TIME (HOURS) 34 urine. as determined by HPLC or EIA in both species over a 48-hour time period. This obser-vation suggests that the drug is extensively metabolized in the body before elimination. After intramuscular administration in dogs (0.49 mg/kg). less than I % of the drug is excreted in the bile and urine. These observa-tions indicate that the kinetics of FK arc not likely to be altered in renal impairment. How-ever. impairment in liver function is likely to result in drug accumulation. thereby necessi-tating adjustments in the FK dosing regimen. The half·life of FK ranges from 4.4 to 8.0 hours. with a mean of 6.1 hours. The metabolic fate of FK has not yet been characterized. The drug may undergo hydro-lytic cleavage and may also be conjugated and demethylated. If the drug is conjugated and the conjugate is excreted in the bile. there is a potential for enterohepatic recirculation of the drug. This may explain the secondary peaks observed in the plasma concentration-v-time curves in dogs after FK administration. Drug InUraCl;on Because immunosuppressants arc com-monly used in combination. drug interactions are of major concern in transplant patients. Although CyA has been reported to inhibit drug-metabolizing enzyme systems.6 steroids arc known to induce the hepatic mixed-function oxidases.1 We studied the effcct of FK on drug-metabolizing enzyme systems. Male Wistar rats weighing between 230 and 250 g were used in this study. The animals were treated with placebo or 0.12 or 1.2 mg/kg of FK intramuscularly for 14 days. On day 15. the animals were anesthetized by VENKATARAMANAN ET AI. ether. and the livers were excised and washed with Tris-KCl buffer (0.02 mol/L Tris·KCI buffer. pH 7.4. 1.15% KCI). Approximately 4 g of the liver was homogenized in 2 vol of Tris-KCI buffer in a polytron homogenizer. and microsomes were prepared according to standard techniques. The protein a:mtent of the microsomes were determined bv the biuret methodS with BSA as the standard. Cyto-chrome P·450 and Cytochrome b5 contents of the microsomal suspensions were determined by the method of Omura and Sato.' NADPH cytochrome c reductase activity was deter· mined by the method of Phillips and Lang-deniO as modified by Gigon et al.II The N-demethylation of ethylmorphine (5 mmol/ L) was determined in an incubation mixture consisting of 5 mmol/L MgCI2, 0.43 mmol/L NADP. 10 mmol/L glucose-6-phosphate. 1.5 enzyme units of g!ucose-6-phosphate deby· drogenase. 50 mmol/L Tris-HCI (pH 7.4), and 2 mg of microsomal protein according to the method of Nash.1l Table I summarizes the result of this study. At a dose of 0.128 mg/kg of FK. there is a significant decrease in the activity of ethyl· morphine N·demethylase activity. At a dose of 1.28 mg/kg, in addition to ethylmorphine N-dcmethylase activity, cytochrome P-450 and cytochrome c reductase activity was also decreased. This indicates that fK has a dose· dependent effect on different components of the hepatic mixed·function oxidase system. CyA also similarly affects the mixcd·funciion oxidase system in the Iiver.6 Thesc observa· tions suggest that the elimination of coadmin-istered drugs may be altered in the presence of FR. Therefore. dosage adjustment of other r.bIe 1. Eff .. of Ch,_lntr.mulICula, Acimillillt,.tiOn of FK on HetNltlc DnI9-Me'lbollzlnt i,."... p~ Cyt __ b"""~ M--.w CytOcIwwM ~ cAecM:tiM N-OInw ....... "-P-411O 110 InrIICIII,*,,- I_HCHOI ~ lmallalu-I t.-J ... "'-i I""., ... ~ rtWtImq """- ....... "-PIICiIbo 24.0 ~ 4.0 0.902 t 0.019 0.488 ~ 0.021 291 :t 65 19.28 t 2.74 0.128 mQlkg 25.8 ~ 1.8 0.825 t 0.074 0.398 t 0.044 267 t 31 13.8S- ~ 2.92 1.28 mQlkg 24.2 t 1.0 0.15715 t 0.0215- 0.412 t 0.022 181 % 17- 6.315- t 1.81 " Sillf1lflclntty off.,.,., II P s .05. iJlOPHARMACEUTlCAL ASPECTS OF FK-506 drugs may be Qecessary. On the other hand. lower doses of other drugs may be effective in patients who receive FK, and this may result in some cost saving. SUMMARY FK is a potent im~unosuppressive agent. fK can be analyzed in biologic fluids by EIA. The oral absorption of FK is rapid but vari-35 able in dogs. After intramuscular administra-tion. FK is slowly and continuously absorbed. FK is primarily eliminated by metabolism. Less than I % of the administered dose is excreted in the bile or the urine. After chronic intramuscular administration FK inhibits drug metabolism. Monitoring of FK levels in plasma is essential for the proper interpreta-tion of efficacy and toxicity studies. REFERENCES 1. Zeevi A: Transplant Proc (this issue) 2. Todo S: Transplant Proc (this issue) 3. Todo S: Transplant Proc (this issue) 4. Todo S: Transplant Proc (this issue) S. Ericzon BG. Todo S, Lynch S, et al: Transplant Proc 19:1248, 1987 6. Augustine JA, Zemaitis MA: Drug Metab Dispos 14:73,1986 7. Shukla V, Garg S. Mathur W: Pharmacology 29: 117. 1984 8. Gornall AG. Bardawill CSt David MM: J Bioi Chern 111:151. 1949 9. Omura T. Sato R: J BioI Chern 239:2310.1964 10. Phillips AH. Langden RG: J BioI Chern 231:2652. 1962 11. Gigon PL, Gram TE. Gillette JR: Mol Pharmacol 5:109.1969 12. Nash I: Biochem J 55:416.1953 ", ,1 ::L t:t ~I " ~t t ::11 " ., ,/ .1 ~ I :1 I I I , .:.::: I . i .. i 

Bioipharmaceutical aspects of FK-506

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Bioipharmaceutical aspects of FK-506

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