Methods of treatment using ADM antibodies

Abstract

The ratio of concentrations of pro-adrenomedullin (pro-ADM)/pro-endothelin (pro-END) immunoreactivity in body fluids of critically ill patients is used as for the diagnosis, course control and prognosis, including an assessment of the mortality risk, of severe life threatening diseases. Further, a treatment of critically ill patients having high levels of pro-ADM but insufficient levels of pro-END immunoreactivities with a medicament comprising vasoconstrictive endothelin or its precursors, and/or endothelin agonists or adrenomedullin antagonists is provided.

Claims

1. A method of treating a critically ill human patient admitted to an intensive care unit (ICU), comprising: determining the concentration, in particular concentration units, of a pro-adrenomedullin (pro-ADM) molecule and/or one or more physiologically occurring fragments thereof using a plasma sample from the patient in an in vitro selective immunodiagnostic assay recognizing a physiologically occurring fragment that is not the mature ADM, determining the concentration in said concentration units, of a pro-endothelin (pro-END) molecule and/or one or more physiologically occurring fragments thereof using a plasma sample from the patient in an in vitro selective immunodiagnostic assay recognizing a physiologically occurring fragment that is not the mature END, wherein the immunodiagnostic assays use combinations of antibodies comprising antibodies against peptides having the amino acid sequences according to SEQ ID NO: 2 or 3 for pro-ADM and SEQ ID NO: 6 or 7 for pro-END, calculating the ratio of the pro-ADM concentration to the pro-END concentration, and when the ratio in said patient is above a cut off value of 90, and when the concentration of pro-ADM is >8 nmol/l or an equivalent value according to the determination method employed, administering to the patient an antibody that binds to ADM and blocks its receptor binding sites, in an amount sufficient to reduce the ratio to below the cut off value.

2. The method according to claim 1, wherein the patient is admitted to an intensive care unit with a diagnosis of one or more of sepsis, SIRS, septic shock, multiple organ failure, infections systemic or local, bacterial infections, heart disease, peritonitis, pancreatitis, local and/or systemic inflammation, meningitis, trauma, ruptured aortic aneurysm, intoxication, endotoxemia, anuria, renal insufficiency, arterial hypertension, pulmonary hypertension, atherosclerosis, cancer, congestive heart failure, cardiovascular disease, coronary artery disease, ischemia, antiarrhythmic effects, cancer comprising colorectal cancer, renal and/or heart failure or organ injury.

3. The method according to claim 1, wherein at least one of the antibodies that specifically binds said physiologically occurring fragment of pro-ADM or pro-END is immobilized onto a solid phase and at least one second antibody specifically binds another part of the same fragment and is used for detection of the fragment attached to the solid phase.

4. The method of claim 1, wherein said antibody that binds to ADM is administered by infusion or injection.

5. The method of claim 1, wherein said antibody that binds to ADM is administered with a pharmaceutically acceptable liquid carrier and one or more of stabilizers, nutrients, electrolytes, dissolved salts, osmotic agents for providing isotonic conditions, buffers and/or solvents and/or other adjuvants and excipients suitable for intravenous administration.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the concentration of ADM-r in plasma of 40 healthy control individuals, 126 surviving patients at ICU and 23 patients, two days and one day before death and on the day of death.

(2) FIG. 2 shows the concentration of END-r in plasma of 40 healthy control individuals, 126 surviving patients at ICU and 23 patients, two days and one day before death and on the day of death.

(3) FIG. 3 shows the ratio of ADM-r/END-r in plasma of 40 healthy control individuals, 126 surviving patients at ICU and 23 patients, two days and one day before death and on the day of death.

(4) FIG. 4 shows the ratio of ADM-r/END-r including only patients having a ADM-r concentration>8 nmol/l in plasma; 24 being surviving patients at ICU and 20 further patients, two days and one day before death and on the day of death.

EXAMPLE 1—PEPTIDE SYNTHESIS

(5) An N-terminal cystein residue was added to synthesized Peptides of human pro-ADM and pro-END. They were purified using mass spectrometry and the quality controlled using reversed phase HPLC and lyophiliysed in aliquots (Jerini AG, Berlin, Germany) according to standard procedures known to the person skilled in the art.

EXAMPLE 2—CONJUGATION AND IMMUNIZATION

(6) Peptides of Sequence IDs 2, 3, 6 and 7 were conjugated to the carrier protein KLH (keyhole limpet hemocyanin) by MBS (Maleimid-obenzoyl-N-hydroxysuccinimid ester) according to the protocols for “NHS-esters-maleimide crosslinkers” by PIERCE, Rockford, Ill., USA. Sheep were immunized receiving 100 μg of conjugate (μg according to the peptide content of the conjugate). Starting at month 4 after immunization every four weeks 700 ml of blood were withdrawn from every sheep and antiserum was gained by centrifugation. Conjugation, immunizations and production of antisera were done by MicroPharm, Carmerthenshire, UK.

EXAMPLE 3—PURIFICATION OF ANTIBODIES

(7) The polyclonal antibodies from sheep were purified using ligand specific affinity purification. For that step the Cys(0)-peptides were linked to SulfoLink-Gel supplied by Pierce (Boston, USA). The binding occurred according to the protocol of Pierce. 5 mg of peptide were added per 5 ml of gel.

(8) In summary, columns were washed three times with 10 ml elution buffer (50 mM citric acid, pH 2.2) and binding buffer (100 mM sodium phosphate, 0.1% Tween, pH 6.8). 100 ml of sheep antiserum were filtered using a filter diameter of 0.2 μm and added to the column material. The gel was eluted quantitatively with 10 ml binding buffer. The material was incubated over night at room temperature by gentle rotation. The material was transferred to empty columns (NAP 25, Pharmacia, emptied). The eluates were discarded. Subsequently the columns were washed with 250 ml protein-free binding buffer (protein content of washed eluate<0.02 A280 nm). Elution buffer was added to the washed columns and fractions of 1 ml were collected. The protein content of each fraction was determined by the BCA-method (according to the protocol of PIERCE, Rockford, Ill., USA). Fractions of a protein content>0.8 mg/ml were pooled.

EXAMPLE 4—TAGGING

(9) 500 μl of affinity purified antibodies generated against the peptides of Sequence IDs 2, 3, 6 and 7 were rebuffered in 1 ml 100 mM potassium phosphate buffer (pH 8.0) was re-buffered via a NAP-5 gel filtration column (Pharmacia) according to the protocol of Pharmacia.

(10) For the tagging with a chemiluminescent marker 10 μl of MA70-Akridinium-NHS-ester (1 mg/ml; Hoechst Behring) were added to 67 μl of antibody solution and incubated for 15 minutes at room temperature. The solution was rebuffered in 1 ml solvent A (50 mM potassium phosphate, 100 mM NaCl, pH 7.4) in a NAP-5 gel filtration column according to the protocols of Pharmacia, particles of low molecular weight were eluted. For elution of unbound labels a gel filtration HPLC was done (Column: Waters Protein Pak SW300). The sample was added and chromatographs at a flow rate of 1 ml/minute in solvent A. The sample was measured at wave length of 280 and 368 nm to determine the degree of tagging. The absorption ratio 368/280 nm had a peak of 1.0. The fractions containing monomeric antibodies were collected (retention time 8-10 minutes) and taken up in 3 ml 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serum albumin, 0.1% sodium azide, pH 7.4).

(11) The tagged antibody was used in a sandwich assay as well as in different assays like SPALT-assays.

EXAMPLE 5—COUPLING

(12) For a sandwich-assay the purified antibodies against the peptides of Sequence IDs 2, 3, 6 and 7 were immobilized on irradiated polystyrol tubes (Greiner, Germany). For that procedure the antibody solutions were diluted to a protein concentration of 6.7 μg/ml with 5.0 mM Tris, 100 mM NaCl, pH 7.8. 300 .mu.l of diluted protein solution per tube were pipetted. These were incubated for 20 hours at room temperature, the solution was removed. Then 4.2 ml of a 10 mM sodium phosphate, 2% Karion FP, 0.3% bovine serum albumin, pH 6.5 solution were added to each tube. After 20 hours the solution was removed and the tubes dried in a vacuum drier. The procedure can also be done as inverse sandwich assay according to procedures known to a person skilled in the art.

EXAMPLE 6—SANDWICH IMMUNOASSAY

(13) The following assay buffer was used: 100 mM sodium phosphate, 150 mM NaCl, 5% bovine serum albumin, 0.1% unspecified sheep IgG, 0.1% sodium azide, pH 7.4.

(14) The protein content of EDTA-plasma of healthy individuals and patients of various diseases/diseases mentioned above was determined.

(15) In the tubes containing the immobilized antibody EDTA-plasma of patients was pipetted (10 μl ADM and 50 μl END-r). 200 μl of PBS (phosphate buffered saline), 0.1% bovine serum albumin were added to each tube and incubated at room temperature for 2 hours. After washing 3 times with 4 ml PBS 200 μl acridiniumester-tagged polyclonal sheep antibody (affinity purified, raised against the peptide 83-94 [Sequence ID 2] and 69-86 [Sequence ID 3] of pre-pro-ADM 1-185 as well as partial peptide 168-181 [RSSEEHLRQTRSET=Sequence ID 6] and 200-212 [SRERYVTHNRAHW=Sequence ID 7] of pre-pro-END 1-212, 20 ng each in PBS, 0.1% bovine serum albumin) respectively. After further incubation of 2 hours at room temperature unbound tagged antibodies are removed by washing 5 times with 2 ml PBS each. Tagged antibody bound to the tube was quantified by measuring the luminescence in a luminometer (Berthold LB 952T/16).

(16) The individual values measured were quantified according to a usual calibration curve that was established using defined dilutions of synthesized pre-pro-ADM 45-92 and pre-pro-END 169-212 as standard solutions.

EXAMPLE 7—MEASUREMENTS

(17) Concentrations of ADM-r and END-r in Healthy Individuals and State of Disease

(18) 149 patients were included in the study who were at the intensive care unit for different reasons and who developed a sepsis during their stay at the intensive care unit (criteria: www.talessin.de/scripte/medizin/sepsis1.html). Every day during their stay at the intensive care unit (3-29 days) EDTA-plasma of the patients was obtained and stored at −20° C. until further use.

(19) ADM-r is present in plasma of healthy individuals at concentrations of <1 nmol/l (median: 0.7). Critically diseased persons who survived a sepsis show a significantly increased ADM-r concentration (median 3.1). The median of dying patients is significantly increased as compared to the group of surviving patients (median 11.8/17.1/17.7; 2 days before exit/one day before exit/on the day of death respectively, FIG. 1).

(20) The concentration of END-r (pro-END) as a putative antagonist of ADM increases from a median of 0.035 nmol/l plasma to 0.054 nmol/l in surviving patients. The concentration further increases to 0.096 and 0.133 nmol/l two days before exit and one day before exit respectively. Surprisingly the concentration of END-r decreases dramatically on the day of death (median 0.064 nmol/l, FIG. 2).

EXAMPLE 8—EVALUATIONS

(21) ADM-r and the Prognosis for Severity of Disease/Disorder

(22) The concentration of ADM-r was used to determine the prognosis of survival (Table 1 and FIG. 1). Column 2 of Table 1 (specificity) shows the number of surviving patients having a concentration of less than 17 nmol/l plasma of ADM-r, i.e. 117 of 126 patients have a concentration of less than 17 nmol/l, 102 patients a concentration of less than 8 nmol/l. Thus ADM-r shows a specificity of 93% for survival at a cut off at 17 nmol/l and 81% at a cut off at 8 nmol/l. The sensitivity of mortality is determined in the following way: 23 patients died during their stay at ICU. At a cut off value of 17 nmol/l of ADM-r six patients (26%=sensitivity of mortality) showed concentrations above 17 nmol/l two days before exit, 12 patients (52%) one day before exit and 13 patients (57%) on the day of death. At a cut off value at 8 nmol/l, 19 (83%) of 23 patients showed concentrations above 8 nmol/l of ADM-r two days before exit, 21 patients (91%) one day before exit and 19 patients (83%) on the day of death (Table 1).

(23) Thus a cut-off value at 8 nmol/l of ADM-r, i.e. excluding the patients having a concentration below 8 nmol/l of ADM-r, includes most of the patients who died during their stay at ICU.

(24) TABLE-US-00001 TABLE 1 Sensitivity Specificity 2nd day- Last day day of Survivals before death before death death n = 126 n = 23 n = 23 n = 23 ADM-r Cut off 17 nmol/l 117 6 12 13 Cut off 8 nmol/l 102 19 21 19 Ratio ADM-r/END-r Cut off 215 117 0 0 14 Cut off 90 96 21 22 20 Ratio ADM-r/END-r ADM-r >8 nmol/l 117 20 21 18 Cut off 90 Sensitivity Specificity 2nd day Last day day of Survivals before death before death death ADM-r Cut off 17 nmol/l 93% 26% 52% 57% Cut off 8 nmol/l 81% 83% 91% 83% Ratio ADM-r/END-r Cut off 215 93%  0%  0% 61% Cut off 90 76% 91% 96% 87% Ratio ADM-r/END-r ADM-r >8 nmol/l 93% 87% 91% 78% Cut off 90

EXAMPLE 9—RATIO

(25) Ratio ADM-r/END-r

(26) The ratio of ADM-r/END-r is shown in Table 1 and FIG. 3. The ADM-r/END-r ratio in healthy individuals has a maximum value of about 50 (median 20). The median of surviving patients at ICU is increased to 56. Significant further increases can be observed two days (median 135) and one day (median 129) before exit and a surprisingly dramatic increase on the day of death at a median of 336. The prognosis at a specificity of 76% (Table 1, i.e. patients included having a concentration of ADM-r of less than 43 nmol/l is 91% two days before exit, 96% one day before exit and 87% on the day of death (Table 1 and FIG. 3). At a higher specificity of 93%, i.e. patients included having a concentration of ADM-r of less than 18 nmol/l, a prognosis is only possible on the day of death having a sensitivity of 61% (Table 1).

EXAMPLE 10—RATIO ADM-R/END-R INCLUDING PATIENTS HAVING A CONCENTRATION OF ADM-R ABOVE 8 NMOL/L

(27) Thus the patients were further selected according to the ADM-concentration (>8 nmol/l) and according to the ratio of ADM-r/END-r (FIG. 4).

(28) It was shown that a high portion of surviving patients with an ADM concentration>8 nmol/l at one day of their stay at the ICU, N=24 had a ratio of less than (N=15) and thus their survival was assured. According to that assumption however also the dying patients would be included with a high probability 2 days before their death (sensitivity 87% two days before exit/91% one day before exit/78% on the day of death). Taking into account the sum of patients included in the study (126 surviving patients) the specificity surprisingly is as high as 93%.

(29) Thus the sequential analysis of ADM-r, END-r and the ratio and or algorithm of ADM-r/END-r at an equally high specificity (93%) surprisingly shows a higher sensitivity for the prognosis (87%/91%/78%) as the analysis of ADM (26%/52%/57%) or ratio (0/0/61) in their own (values 2 days before death/one day before death/day of death respectively).