Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

• This invention is directed to the discovery that both the nephrotoxic effect of in-proc, ass nephrotoxic therapy on an individual as well as the potential for nephrotoxic effect of contemplated therapy on a given individual can be evaluated as a function of urine kallikrein content.
• the urine kallikrein level of a patient receiving such therapy is monitored and compared to the patient's baseline (pretherapy) urine kallikrein level.
• low baseline urine kallikrein is predictive of patients who are at greater risk for developing nephrotoxic reaction to therapeutic compositions, such as those comprising cyclosporine.
• Cyclosporine is a cyclic, nonwater-soluble, highly nonpolar molecule composed of eleven amino-acids.
• the compound is a promising immunosuppressive agent which is derived from soil fungus (Calne et al., Transplant Proc. 13:349-358, (1981)? Ferguson et al., Surgery, 92:175-182, (1982); Starzl et al., Gynecol. Obstet., 151:17-26, (1980)).
• the drug is now widely used for prolonging the function of various transplanted organs. Its immunosuppressive effects selectively inhibit T-cell function, allowing survival of allografts without myelosuppression, i.e. heart transplants, Myers et al., N. Eng J. Med. 311:699 (1984).
• Cyclosporine is a lipophilic molecule with a molecular weight of 1202 daltons.
• the drug When the drug is dissolved in olive oil or a special solution prepared by the manufacturer, bioavailability and absorption are maximized.
• the drug readily binds to plasma proteins and has a terminal half-time of 24 hours. It is highly metabolized in the liver, with biliary excretion being the major route of elimination (Beveridge, T., Cyclosporine A:, Proceedings of the International Symposium, Cambridge, White, D.J., ed., pages 35-44 (1982)).
• the drug In addition to its immunosuppressive characteristics, the drug also has interesting anti-schistosome and anti-malarial activities (Kolata, Science (Washington, D.C.) 221:40-42, (1983); Sanches et al., First Int'l. Montreux Conf. on Biol. Rhythms and Medications, Montreux, Switzerland, March 26-30, 1984. Pergamon Press, Oxford, (in press).
• Urinary enzyme analysis provides an extremely sensitive indicator of renal injury.
• Urinary enzyme levels have been shown to be elevated in a wide spectrum of renal diseases including glomerular and interstitial as well as tubular disease, often prior to the onset of renal failure or even before any abnormality in excretory function is detectable, Price, R.G., Toxicology, 23: 99-134 (1982); Maruhn, D., Curr. Probl. Clin. Biochem., 9:135-149 (1979); Piperno, E., supra, pp. 31-55; Sherman, R.L. et al., Arch. Intern. Med., 143:1183-1185 (1983).
• N-acetyl-beta-D-glucosaminidase a lysosomal enzyme present in renal tubular cells
• Kallikrein is an endopeptidase that generates vasoactive polypeptides from kininogens, plasma alphas-globulin substrates (Werle, E. et al., Sub- stanz. Biochem. Z., 289:217 (1937)). It is secreted as an inactive precursor which can be activated in vitro by trypsin (Spragg, Adv. Exp. Med. Biol., 156A:393 (1983)) or thermolysin (Noda et al., Kidney Int., 27:630-635 (1985)).
• Human urinary kallikrein (urokallikrein or HUK) , when purified on the basis of its kinin-generating activity by conventional chromatographic techniques, has been shown to contain a non-kinin-generating alkaline TAMe esterase which can be separated from urokallikrein by broad range isoelectric focusing or by alkaline polyacrylamide disc gel electrophoreses (ole-MoiYoi, o. et al., Biochem. Pharmacol. 26:1893 (1977)). Electrophoretic, antigenic, and immunohistochemical studies have established that urokallikrein and the TAMe esterase represent two distinct renal enzymes (Pinkus, G.S. et al., J. Histochem.
• a need had continued to exist for a non-invasive means for evaluating cyclosporine-related nephrotoxicity and a means for predicting potential high-risk cyclosporine therapy patients.
• a need has continued to exist for an non-invasive means for evaluating the nephrotoxic effect of other potentially nephrotoxic agents.
• a need had also continued to exist for a means for distinguishing between nephrotoxicity and allograft rejection in patients being treated with cyclosporine. Summary of the Invention.
• the present inventors first succeeded in correlating urinary kallikrein levels with cyclosporinerelated nephrotoxicity. It was thus realized that kallikrein levels are an indication of the nephrotoxicity of cyclosporine and of other potentially nephrotoxic compositions.
• total urinary kallikrein levels are measured utilizing a radioimmunoassay for human urokallikrein, said radioimmunoassay comprising a monospecific antibody which has been shown to recognize both the active and trypsin-activatable forms of the enzyme (PinKus et al., J. Histochem. Cytochem, 31:1279-1288 (1983)) and a radioligand purified so as to maintain its active site.
• comparison of the rate of urinary kallikrein antigen excretion of the cyclosporine-treated patient with pre-treatment base line levels of urine kallikrein provides a means for evaluating the nephrotoxic effect of the cyclosporine therapy.
• the same method is applicable to other nephrotoxic agents.
• total urinary kallikrein levels are measured as above and compared to the patient's prior kallikrein levels dur ing cyclosporine treatment.
• the nephrotoxic effect of cyclosporine may be monitored. For example, decreases in kallikrein will reflect impaired renal function and indicate that cyclosporine administration should be reduced or terminated.
• Increases in kallikrein reflect improved renal function and indicate that cyclosporine administation may be increased or maintained.
• Steady kallikrein levels indicate that cyclosporine administration may be maintained.
• the same method is applicable to other nephrotoxic agents.
• increases in serum creatinine in a patient undergoing therapy with agents which are potentially nephrotoxic may be predicted by measuring kallikrein levels during cyclosporine treatment. It has been found that episodic decreases in kallikrein levels are temporally related to increases in serum creatinine. Thus, kallikrein decreases presage serum creatinine increases by one to four months. This method provides an early and sensitive indicator of renal dysfuction brought about by cyclosporine treatment. The same method is applicable to other nephrotoxic agents.
• measurement of urinary kallikrein in pre-therapy patients provides a means for predicting potential nephrotoxic response to cyclosporine or other nephrotoxic therapy.
• Figure 1 is a graphic representation of the effect of cyclosporine on urinary kallikrein (HUK) excretion in patients with rheumatoid arthritis. Drug administration was begun at week 1 and terminated at week 24. Patients represented here had normal or elevated control excretion rates. Means and standard deviations are shown for the weeks completed by all patients. The asterisks in Figure 1 indicate that the mean for weeks 12 and 24 were significantly different from the base line value (p ⁇ 0.01). Because of the high base line HUK excretion rate of patient 1, her subsequent values were not included in the statistical analysis.
• HUK urinary kallikrein
• Figure 2 is a graphic representation of the effects of cyclosporine administration on urinary kallikrein excretion rates in rheumatoid arthritis patients who had significantly depressed HUK excretion baseline levels.
• the drug schedule was as described in Figure 1. Patients 5 and 10 were dropped from the study. Description of the Preferred Embodiments.
• kallikrein excretion was measured in patients with rheumatoid arthritis who were participating in a trial to evaluate the efficacy of cyclosporine therapy.
• kidney damage may lead to the urinary excretion of enzymes other than kallikrein that are capable of cleaving the frequently used tripeptide p-nitroanilide substrate S-2266 (Koolen, M. I. et al., Transplantation, 37:471-474 (1984)), and because urine contains esterases other than kallikrein (Levinsky, N. G. et al., supra), the excretion of urokallikrein must be measured utilizing an assay which takes the above into account.
• a suitable assay is the active site radioimmunoassay of Silver et al., supra.
• a similar radioimmunoassay to measure total kallikrein antigen without the necessity of trypsin treatment of urine samples employs an antibody which recognizes both active and latent forms of kallikrein and therefore measures total kallikrein directly (Pinkus et al., J. Histochem. Cytochem., 31:1279-1288 (1983)).
• Urokallikrein Human urokallikrein for use in the radioimmunoassay is purified from concentrated fresh pooled human urine, typically by affinity chromatography modified for a batch procedure and by gel filtration. Concentrated urine is incubated with gel. The affinity gel is then washed and the urokallikrein is eluted from the affinity gel and collected. The urokallikrein is further purified, i.e., by gel filtration as described, and the final preparation is purified to homogeneity.
• the purified urokallikrein is then labeled using techniques known to the art.
• Typical labels include radiolabels, enzyme labels, chemiluminescent labels, fluorescent labels, free radical labels and the like. Radiolabeling is preferred, with a typical labeling of urokallikrein effected as below.
• 125 I-monoiodo-Bolton-Hunter reagent is mixed with purified urokallikrein and then incubated overnight.
• reaction mixture is applied to an equilibrated
• the monospecific anti-urokallikrein IgG used for the active site specific radioimmunoassay inhibits both the kinin-generating and esterolytic functions of purified urokallikrein in a dose-dependent fashion, thereby indicating specificity for determinants at or near the active site of the enzyme.
• the monospecific anti-urokallikrein IgG used for the total kallikrein radioimmunoassay does not inhibit the interaction of kallikrein with the small ester substrate (Pinkus et al., J. Histochem. Cytochem., 31:1279-1228 (1983)).
• IgG fractions of both anti-sera were prepared by chromatography, typically DE-52 cellulose, and filtration, typically Sephadex (ole-MoiYoi et al., J. Immunol., 121:66-71 (1978)).
• the radioimmunoassay is performed in sodium borate buffer (i.e., 0.1 M) containing gelatin. Portions of a 1/400 dilution of anti-urokallikrein IgG are mixed with crude urine samples or with dilutions of urokallikrein standard in buffer; the final volume of each reaction mixture is made equal with buffer. After the addition of 125 I-urokallikrein, the samples are incubated.
• An antibody titration curve developed by using varying doses of anti-urokallikrein IgG and a fixed amount of 125 I-urokallikrein, is dose-related between dilutions of anti-urokallikrein.
• concentrations of urokallikrein standard i.e., 0.2 to 3 ng/20 ul
• the ratios of bound to free counts are plotted as a function of the concentration of urokallikrein standard.
• the amount of 125 I-urokallikrein used is chosen such that approximately 80% of the bindable counts are precipitated by the 1/400 dilution of anti-urokallikrein IgG used for the radioimmunoassay. Crude urine samples are assayed in duplicate in 5- to 30-ul volumes and bound: free count ratios are used to determine urokallikrein concentrations from the standard curve.
• Urine samples are immediately placed at 4°C and are assayed on the day of collection or frozen at -70°C until they are assayed.
• urine samples are exposed to insolubilized trypsin.
• Trypsin-CH-Sepharose 4B is prepared by using activated CH-Sepharose 4B in a modification of the manufacturer's recommended procedure.
• the activated-CH-Sepharose 4B is washed in a coarse Buchner fritted glass funnel with ice-cold 1 mM HCl, followed by potassium phosphate, pH 5.8.
• the gel is then transferred to a solution of the same buffer containing trypsin.
• the unreacted trypsin is separated from the trypsin-CH-Sepharose 4B and the gel is treated with ethanolamine and washed with buffers and stored.
• Urine samples are treated with trypsin by mixing insolubilized trypsin with cold urine and incubating at room temperature with shaking. The tubes are then centrifuged and the supernatants are removed and stored until assayed.
• the enzymatic activity of urokallikrein is assessed by its ability to generate kinin from heatinactivated human plasma.
• Samples of urine, with or without exposure to insolubilized trypsin are incubated with heat-inactivated plasma.
• the mixtures are immediately assayed for formed kinin on a guinea pig terminal ileum or estrous rat uterus segment.
• the kinin generated is quantitated by comparison with the contractile response of the tissue to standards of synthetic bradykinin.
• the present invention is dependent upon an assay for kallikrein which is sufficiently sensitive to detect changes in levels of excretion of kallikrein while avoiding spurious data resulting from the activity of enzymes other than kallikrein.
• the assay also avoids spurious data resulting from measurement of other enzyme products (peptide) which may be active in the bioassays.
• the assay is technically feasible and has the particular advantage that procurement of the assay sample (urine) does not
• urinary kallikrein is determined in a patient prior to the inception of cyclosporine therapy. In this manner, the patient's baseline (pre-therapy) level of kallikrein is established. Once the baseline value of the kallikrein excretion is established, this value provides two functions.
• subsequent kallikrein excretion level determinations such determinations made subsequent to the inception of, and during the course of, cyclosporine therapy, may be compared to the baseline level of kallikrein excretion. The comparison then provides a measure of the degree to which the cyclosporine therapy is impacting upon kidney function, i.e., the degree of nephrotoxicity resulting from the cyclosporine therapy.
• this baseline value provides a predictive function.
• the baseline level for a particular patient indicates levels of kallikrein excretion which are substantially lower than the average baseline levels for a normal control population, i.e., 113.68 + 8.39 ng/24 hr. (see Table 2) then one can quite accurately predict that the proposed cyclosporine therapy is contra-indicated.
• urinary kallikrein is determined in a patient during the course of cyclosporine therapy.
• the frequency of urinary kallikrein determinations can be varied to suit the particular needs of the patient in view of the patient's medical history, age, health, etc., and the many other variables affecting such determinations, as is well known to those of skill.
• Monthly determinations are preferred, but determinations at more or less frequent intervals may be indicated in a particular case.
• a decrease in kallikrein will indicate that cyclosporine administration should be decreased or stopped to avoid damage to the kidneys.
• kallikrein levels remain stable during the course of cyclosporine therapy, this indicates that renal function is not degenerating.
• the onset of cyclosporine treatment often will result in a change in kallikrein levels that reflects the nephrotoxic effect of the drug. Assuming that the treatment level is therapeutically acceptable, however, maintenance of stable kallikrein levels indicates that cyclosporine administration may safely be maintained.
• an increase in kallikrein levels during ongoing cyclosporine treatment suggests improved renal function. This indicates that cyclosporine administration may be increased.
• this embodiment of the present invention does not rely upon comparisons of a patient's kallikrein levels with that patient's pre-treatment baseline levels or with a statistically derived standard baseline level. Instead, the patient's own kallikrein levels are monitored during cyclosporine treatment and compared with prior levels during the same course of treatment.
• This embodiment of the invention provides a means by which nephrotoxic effect may be predicted at a given cyclosporine dosage, and the dosage adjusted to preclude renal damage. It also provides a means by which the therapist may optimize cyclosporine administration during the treatment of an individual patient, by monitoring that patient's kallikrein levels.
• decreases in urinary kallikrein provide a means of predicting subsequent increases in serum creatinine.
• patients receiving chronic cyclosporine therapy at reduced doses (3 mg/kg/day) were observed to experience episodic decreases in kallikrein excretion.
• the invention has utility for evaluating the nephrotoxic effect of other agents, such as aminoglycoside antibiotics, analgesic drugs, i.e., phenacetin, non-steroidal anti-inflammatory drugs, e.g., aspirin, and environmental contaminants as described above, as well.
• agents such as aminoglycoside antibiotics, analgesic drugs, i.e., phenacetin, non-steroidal anti-inflammatory drugs, e.g., aspirin, and environmental contaminants as described above, as well.
• NSAIDS nonsteroidal antiinflammatory drugs
• prednisone at doses of less than 10 mg/day were continued (Table 1).
• Patient 10 was also taking a beta-blocker (Corgard). Gold salts, D-pencillamine, or methotrexate were discontinued at least 2 months prior to the study.
• Cyclosporine was administered for a period of 24 weeks, followed by a 12-week washout phase.
• Renal function was clinically evaluated by monitoring serum creatinine and BUN, and serious nephrotoxicity which required interruption of the protocol was defined as a serum creatinine of greater than 2.0 mg/100 ml or a BUN of greater than 40.0 mg/100 ml.
• Urinary kallikrein excretion does not appear to show a consistent or significant pattern of diurnal variation in normal individuals (Table 2). This finding made it possible to request that patients collect urine for 12 hours (rather than 24 hr.) in order to enhance compliance. Collections were made at weeks 0, 12, 24, 28, 32 and 36. Urine was stored at 4°c during the collection period and brought the next day to the Ambulatory Center. An aliquot was removed and frozen at -70°C for kallikrein radioimmunoassay and the rest was submitted for determination of sodium, potassium, creatinine, and protein.
• creatinine clearance was calcu lated directly using the data obtained from the 12-hr urine sample; creatinine clearance was also estimated as described by Gates, G. F., Am. J. Kidney Diseases, 5:199-205 (1985).
• Total urinary kallikrein was assayed as described by radioimmunoassay (Spragg, J. et al., Kidney Internat., 28:75-81 (1985), Silver, M. R. et al., J. Immunol., 124:1551-1555 (1980)), but with an antiserum that is not specific for epitopes expressed only on the active form of kallikrein (Pinkus, G.S.
• the group of four patients with low baseline kallikrein excretion rates included the two individuals in whom cyclosporine administration was terminated.
• Patient 10 demonstrated significant neph- rotoxicity by week 4 as indicated by a BUN of 45 mg/100 ml.
• cyclosporine was administered for 24 weeks, followed by a 12-week washout period. Renal function was evaluated by serum creatinine and BUN, and serious nephrotoxicity that required interruption of the protocol was arbitrarily defined as a serum creatinine greater than or equal to 2.0 mg/100 ml or a BUN greater than or equal to 40.0 mg/100 ml.
• phase two The temporal relationship between decreased kallikrein excretion and increased serum creatinine was assessed by monthly sampling while patients received a reduced dose of cyclosporine (phase two). Of the seven patients participating in phase two, five patients had episodes during which kallikrein excretion decreased by 49 ⁇ 15% over a one-month interval with no increment in serum creatinine (Table 4). When the observation period was extended to two months, the percent decrease in kallikrein from the beginning of the episodes was 72 ⁇ 13% and increments in serum creatinine were associated with three of the episodes. When the observation period was extended to four months, serum creatinine elevations were seen in the rest of the patients in association with a maintained depression in kallikrein excretion. The occurrence of these episodes was not particularly associated with low or normal baseline kallikrein excretion rates.
• the present invention provides a novel method for detecting and evaluating nephrotoxicity in response to potentially nephrotoxic
• nephrotoxic chemicals such as cyclosporine, aminoglycoside antibiotics, analgesics such as phenacetin, and non-steroidal antiinflammatory agents such as aspirin, as well as a multitude of nephrotoxic chemicals found in our environment. While monitoring serum creatinine provides information relating to filtration function, and monitoring BUN reflects both glomerular filtration and sodium-associated tubular readsorption, kallikrein excretion provides a measure of the synthetic and secretory function of the distal nephron, which may be significantly altered in certain circumstances where filtration/readsorption is within normal limits.
• radioimmunoassays include heterogeneous or solid phase radioimmunoassay, single antibody methods or “double" antibody methods, and direct (forward) or reverse sandwich assays.
• a typical solid phase system includes antibody covalently coupled to an insoluble support so that both the antibody and the bound complex after incubation can be readily separated from the soluble free fraction.
• solid phase supports include particles of dex stran, cellulose, continuous surfaces such as polystyrene or polypropylene disks, walls of plastic tubes, glass disks, glass particles, and the like. Particular solid phases are widely used for a variety of different assays and are included in the present invention.
• Another known method for immobilization involves the biotin-avidin complex formation.
• Enzyme immunoassays may be used as well. In this technique, enzymes are applied labels on antigen or antibodies for identification and localization of the immunoreactants. Any method in which the extent of binding of enzyme-labeled antigen, or enzyme labeledantibody to its immunoreactant is measured, is included in this invention. Enzyme immunoassays may be classified as homogeneous or heterogeneous, depending on whether the labeled reagent behaves differently or identically whether or not it is bound to specific counterparts in the immunoreaction, in which therefore it may or may not require physical separation of the reactants into two fractions.
• Another immunoassay method in the present invention is the latex agglutination method.
• latex particles are coated with antigen and incubated with a high affinity hybridomally produced antibody. Inhibition of agglutination will recur when a sample of physiological fluid containing the antigen TABLE 1: Characteristics of rheuastold arthritis patients enrolled in the cyclosporine (CsA) protocol
• agglutination is incubated with this mixture.
• the inhibition of agglutination can either be followed with a counter or by infrared absorption techiques.

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