Iqbal, M P

Malik, I

Mehboobali, N

English

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eCommons@AKUDepartment of Biological & Biomedical Sciences Medical College, PakistanAugust 1992Appearance of a methotrexate binding plasmaprotein during high dose methotrexate therapyM P. IqbalAga Khan University, perwaiz.iqbal@aku.eduI MalikAga Khan UniversityN MehboobaliAga Khan University, naseema.mehboobali@aku.eduFollow this and additional works at: https://ecommons.aku.edu/pakistan_fhs_mc_bbsPart of the Biochemistry Commons, and the Medicine and Health Sciences CommonsRecommended CitationIqbal, M. P., Malik, I., Mehboobali, N. (1992). Appearance of a methotrexate binding plasma protein during high dose methotrexatetherapy. Journal of Pakistan Medical Association, 42(8), 193-196.Available at: https://ecommons.aku.edu/pakistan_fhs_mc_bbs/402APPEARANCE OF A METHOTREXATE BINDING PLASMAPROTEIN DURING HIGH DOSE METHOTREXATETHERAPYPages with reference to book, From 193 To 196 M. Perwaiz lqbal, Nasima Mehboobali  ( Departments of Biochemistry, The Aga Khan University Medical College,Karachi. ) Imtiaz Malik  ( Departments of Medicine, The Aga Khan University Medical College, Karachi. ) INTRODUCTION Methotrexate (MTX) is one of the most widely used oncocytotopic drugs1. Recently it has been used inextremely high doses (8-15 gms/m2) in the treatment of osteosarcoma2, non-Hodgkin’s lymphoma andmetastatic head and neck cancer3. Such high doses facilitate the passive entry of MTX in most solidtumours and thereby provide the necessary tumour cell kill. However, to achieve the desired effects ofthis treatment, it is usually given repeatedly over extended periods of time depending upon the type ofregimen being followed. Toxicity of the drug is prevented by ensuring its rapid clearance from the bodyby proper hydration, alkalinization and restoring to leucovorin "rescue"4. However, reports of delayedplasma clearance of the drug during the course of therapy in certain patients with osteosarcoma due toone reason or another and the ensuing toxicity are not very uncommon5,6. We report a patient sufferingfrom osteosarcoma who developed severe toxicity following third infusion of MDC due to delayedplasma clearance of the drug.CASE REPORT A 19 year old male was admitted to The Aga Khan University Hospital with pain and tenderness in theleft knee. Incisional biopsy from the lower end of the femur established the diagnosis of osteogenicsarcoma. Radiological examination of liver and chest indicated no evidence of metastasis. The patientwas put on chemotherapy and was first given a course with cyclophosphamide- bleomycin-adriamycinand then two weeks later put on high dose MTX therapy (12 gm/m2 every week). In every cycle oftreatment, MTX (18.7 gm) was administered in the form of 6-hr infusion. This therapy wasdiscontinued after the third MDC infusion as he developed symptoms of severe toxicity which includedfever, chills, nausea, vomiting, maceration of skin, erythema, pancytopenia, etc. During chemotherapy,serum creatinine levels remained almost within the normal range. MIX levels in plasma weremonitored by a very sensitive radioassay7,8. Leucovorin ‘rescue’ (15 mg/rn2 every6hr) was started 24hours post MIX infusion and continued till the levels of MTX dropped below 1x107 M.Experimental methodsA. Immuno-precipitation of plasma proteins: In order to determine that the circulating MTX was boundto which type of plasma protein, 0.5 ml of patients plasma sample obtained 9 days following the secondinfusion of MDC was dialyzed extensively against normal saline at 4°C for 24 hours. Twenty five ulfrom the dialyzed plasma was used to set up each immune reaction. Three different immune reactionswere set up each containing 25 ul of dialyzed plasma and 225 ul of 0.006M citrate buffer, pH 7.4. Then250 ul of anti-human albumin or anti-human a1 acid glycoprotein or anti-human IgG was added torespective tubes. Appropriate control reactions were also set up. These contained 25 ul of normal salinewith free MDC equal to the amount in the dialyzed patient’s plasma, 250u1 of antibody to a plasmaprotein (anti-human albumin or anti-a1 acid glycoprotein or anti-human IgG) in a total volume of 0.5ml made up with 0.006 M citrate buffer, pH 7.4. All reactions including controls were incubated at 4°Cfor 48 hours and then centrifuged at 3,000 rpm at 4°C for 15 min. The supernatant solutions werediscarded and the precipitate in each tube was quickly washed with 0.5 ml of cold saline. Theprecipitate was then dispersed in 250/41 of 0.006M citrate, p11 7.4, and boiled for 5 mi to extract MDCwhich was then assayed by the radioassay7,8.Gel-filtration of patient’s plasmaOne ml of patient’s plasma obtained 30 hours after the third MDC infusion was applied to a column(1.5 cmx75 cm) of sephadex G-75 as described earlier9. The sample was eluted with 0.05 M Tris-HClbuffer, pH 7.2 at a flow rate of 12 mI/hr. One ml fractions were collected and MIX in every otherfraction was monitored by the radioassay. Dcxtran blue, human serum albumin and 3H2o were used ascolumn markers in a separate run on the gel.Additional binding of [3H]MTXSince most plasma proteins are non-specific binders of MDC additional binding of [ H]MTX at pH 7.2by patient’s plasma was carried out according to the method described previously10 with the differencethat 50/41 plasma was used as binder instead of L1210 cytosol and incubation of the reactions wascarried out for 1 hour at 37°C.Serum electrophoresisElectrophoresis of two serum samples of the patient before and after second MDC infusion was carriedout according to Beckman Paragon Electrophoresis system SPE (Beckman Instruments Inc., Brea, CA).The agarose gel after the electrophoresis was dried into a film which was then scanned in a Beckman(Appraise) densitometer.RESULTS AND DISCUSSION Figure 1 shows the decay curves of plasma MTX after 6 hr infusions (18.7 gms MTX in each infusion).The elimination of the drug during all three infusions appears to be biphasic during the first 48 hr.Initial phase of elimination half-lives (t1/2a) during the first and second infusions was calculated to be1.5 hr - 2 hr while the second phase of elimination (t1/2B) was found to be approximately 5 hr. .6 hr.These half-life values were not much different from those reported by us previously and by otherinvestigators11-13. However, during the third infusion, there was a substantial change in eliminationhalf-lives of the drug. The t1/2a value was 3.5 hr - 4 hr while t1/2B value turned was more than 12 hrsince binding of a drug to plasma proteins has been known to significantly influence, itspharmacodynamic properties and pharmacokinetic parameters, this slower plasma clearance of MTXfollowing third infusion was attributed to be due to its binding to a plasma protein14,15. This binding ofthe drug to the plasma protein must be strong enough so that it does not come off very easily. Differentsamples of the plasma taken at different time intervals following MDC infusions were analyzed fortheir ability to bind [3HJMTX.Figure 2 shows additional binding of [3H]MTX by patient’s plasma before and after first, second andthird MDC infusions, it is evident that following second infusion, there appears something in plasmathat can bind [3]MTX more avidly and this binder of MDC persists in plasma even 16 days post thirdinfusion. To find out the exact nature of this binder of MTX, patient’s plasma obtained after secondinfusion was extensively dialyzed against normal saline and then treated with anti-human IgG, anti-human a1 acid glycoprotein and anti-human albumin in separate reactions.Table shows that the bound MTX in patient’s plasma was complexed with albumin. Furtherconfirmation of this observation was obtained by subjecting patient’s plasma (following third course ofMTX therapy) to Sephadex G-75 gel- filtration chromatography (Figure 3).There is a single peak of MDC bound to a protein which appears to have a size nearly that of humanserum albumin. Immunoprecipitation of 0.5 ml of fraction 65 (in the peak region) with anti-humanalbumin (1 ml) revealed MTX-albumin complexes in the peak (solid bar in Figure 3) lending furthersupport to the notion that it is the albumin to which MDC has been bound in patient’s serum. Non-specific binding of MDC to human serum albumin has been known for quite some time. However thisbinding is quite weak with two binding constants, 8.88 mM-1 and 17.6mM-1 respectively15. Thebinding of MDC to albumin in patient’s plasma following second infusion is relatively stronger as asignificant portion of the drug did not come off during dialysis of the sample (Table). Moreover,[3H]MTX bound to it did not come off with activated charcoal used in our radioassay (Figure 2). Weare not sure whether this increased binding of MTX to patient’s plasma is due to induction of albuminwith high affinity for MDC or modification of a fraction of albumin to a high affinity form, however,serum proteins profiles of the patient’s serum samples (before and after the second infusion) followingelectrophoresis (Figure 4)show that there is hardly any difference in the levels of albumin in these two samples. It is the propertyof a non-specific binder that it binds more of a drug at higher concentrations of the drug in the reaction.The binding of MDC to albumin before as well as after second infusion of the drug is of non-specificnature.Figure 5 shows that I3H]IMTX binding by the two plasma samples progressively increases as MDCconcentration in the reaction is increased from 13.75 nM-110nM. This indicates that albumin present inplasma before second infusion and after second infusion binds MDC non-specifically, though there isincreased capacity of the second sample (after second infusion) to bind MDC and this perhaps is thereason for delayed plasma clearance of MDC in this patient In general, delayed clearance of the drughas been considered to be due to compromised renal function13. This report suggests that in certainpatients during the course of treatment, certain plasma proteins may undergo a change leading toincreased binding of MDC to plasma. This would delay the clearance of the drug resulting into severeclinical toxicity.ACKNOWLEDGEMENTS We gratefully acknowledge the valuable suggestions by Prof. M. Anwar Waqar in improving themanuscript of the paper. We also appreciate the help rendered by Drs. Amjad Ali and Adnan Siddiqui incollecting clinical samples.REFERENCES 1. Borsi, 3D. and Moe, P.J. A comparative study on the pharmacokinetics of methotrexate in a doserange of 0.5g to 33.6g/m in children with acute lymphoblastic leukaemia Cancer, 1987;60:5-13.2. Grem, J.L., King, S.A., Wines, RE. and Leyland-Jones, B. The role of metbotrcaate in osteosarcoma.JNCl., 1988;80:626-543. Jolivet, J., Cowan, K.H., Curt, G.A., Clendeninn, NJ. and Chabner, B.A. The pharmacology anddinical use Of methotrexate. N. Engl. J. Med., 1983;309:1094-1104.4. Nirenberg. A., Mosende, C., Mehts, B. et al. High-dose methotrexate with citrovorum factor fescue:predictive valUe of serum mçthotrexate concentration and corrective measures to avert toxicity. CancerTreat. Rep., .1977;61:779-83.5. daCosta, M., Isacoff, W., Rothenherg, S.P. and lqbal, M.P. Protein-methotrexate-IgG complexes inthe serum of patients receiving high-dose antifolate therapy. Cancer, 1980;46:471-74.6. Breithaupt, H. and Kuenzlcn, E. Pharmacokinetics of methotrexate and 7-hydroxymethotrexatefollowing infusion of high-dose methotrexate. Cancer Treat. Rep., 1982;66: 1733-417. Rothenberg. S.P., dsCosta, M. and lqbal, MR. Ligand-bindiing rsdiosssay for the antifolatecompounds; application in patients receiving methotrexate. Cancer Treat Rep., 1977;61:575-83.8. lqbal, M.P., Khursheed, M., Mehboobali, N. and Waqar, M.A. Therapeutic monitoring of sntifolatedrugs in biological fluids, Pakistan). PharmacoL, 1988;5: 11-23.9. Iqbal, M.P., Mehboobali, N., Waqar, M.A. and Ahmed, M. Heterogeneity of methotrexate bindinginhuman colon tumourcells. J.Pak.Med.Assoc., 1991;41:136-39.10. Iqbal, M.P., Rothenberg, S.P. and daCoata, M. Evidence for kinetic and immunologic heterogeneityof dihydrofolate reductase in L1210 leukaemia cells. Biochem. Med. Metab. Riot, 1991;46:196-207.11. Iqbal, M.P., Khursheed, M. and Mehboobali, N. Methotrexate clearance and clinical toxicity inosteosarcoma followinghigh dose methotrexate therapy. J.Pak. Med. Asaoc., 1989;39:38-41.12. Pratt, GB., Robert, D., Shanka, E. and Warmeth, EL Response, toxicity and pharmacokinetics ofhigh-dose methotrexate 9NSC-740) with citrovorum factor (NSC-3590) rescue for children withosteosarcoma and other malignant tumoura. Cancer Chemother. Rep., 1975;6:13-18.13. Stoller, R.G., Jacobs, S.A., Drakee, J.C. et al. Pharmacokinetica of high-dos methotrexate (NCS-740). Cancer Chemother. Rep., 1975;6:19-24.14. Imamurs, Y., Kojima, Y. and Ichibagase, H. Effect of simultaneous administration of drugs-onahsorptionand excretion. Binding of acetohexamide and its majormetabolite, (-) hydroxyheamide tohuman serum albumin. Chem. Pharm. Bull., 1985;33:1281-84.15. Skibinska, L., Ramleu, C., Zaluski,J. and Olejniezak, B. Methotreate bindingto human plasmaproteins. Pol.J. Pharmacol. Pharm., 1990;42:151-58.16. Isacoff, W.H., Morrison, P.F., Aroesty, J., Willia, K.L, Block, J.B. and Lincoln, T.LPharmacokinetics of high-dose MTX with rescue as a guide to toxicity and therapy. Cancer Treat.Rep.,1977;61:1665-71.17. Abelson, H.T., Fosburt, M.T., Beardsley, OP., Ooorin, AM., Gorka, C., Link, M. and Unk, D.Methotrexate-induced renal impairment; clinical studies and rescue from systemic toxicity with high-dose leucovorin and thymidine. J.Clin.Oncol., 1983;1:208-16.

Appearance of a methotrexate binding plasma protein during high dose methotrexate therapy

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Appearance of a methotrexate binding plasma protein during high dose methotrexate therapy

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