Combination of anti-FGFR4-antibody and bile acid sequestrant

Abstract

The present invention relates to the amelioration of toxic effects that are caused by therapies aimed at FGFR4-inhibition. In particular, the invention relates to the combination of FGFR4 inhibitors and agents capable of reducing bile acid levels and to the use thereof in the treatment of diseases.

Claims

1. A method for treating hepatocellular carcinoma, said method comprising administrating an effective amount of an FGFR4 inhibitor combination comprising an anti-FGFR4 antibody in combination with colestyramine to a subject in need thereof, wherein the anti-FGFR4 antibody comprises a heavy chain comprising a CDRH1 as shown in SEQ ID NO: 3, a CDRH2 as shown in SEQ ID NO: 8 and a CDRH3 as shown in SEQ ID NO: 15 and a light chain comprising a CORL 1 as shown in SEQ ID NO: 23, a CDRL2 as shown in SEQ ID NO: 25 and a CDRL3 as shown in SEQ ID NO: 30.

2. The method of claim 1, further comprising administering one or more fat-soluble vitamins to the subject.

3. The method of claim 1, wherein the anti-FGFR4 antibody and the colestyramine are administered together or separately, wherein the anti-FGFR4 antibody is intravenously administered and the colestyramine is orally administered.

4. The method of claim 1, wherein the anti-FGFR4 antibody and the colestyramine are administered in a single dose or divided in two or three doses per day.

5. The method of claim 1, wherein the anti-FGFR4 antibody and the colestyramine are administered separately and wherein the anti-FGFR4 antibody is administered in a single dose and the colestyramine is administered in two or three doses per day.

Description

FIGURES

(1) FIG. 1: Serum levels of bilirubin, γ-glutamyltransferase and alkaline phosphatase. The results are shown for administration of U4-3 alone (.circle-solid.), U4-3 in combination with Ursofalk™ (.square-solid.) and U4-3 in combination with Quantalan™ (.box-tangle-solidup.). Dotted vertical lines: U4-3 administration, palliative therapy started with U4-3 on day 0, first measurement after administration was on day 1.

(2) FIGS. 2a-2b: Alaninaminotransferase (ALA T) in serum of cynomolgus monkeys. Upper limit of normal (ULN): 81 UI/I. The results are shown for male monkeys in FIG. 2a and for female monkeys in FIG. 2b. The values shown as .circle-solid. represent administration of U4-3 alone, .square-solid. represents U4-3 in combination with Ursofalk™ and .box-tangle-solidup. represents U4-3 in combination with Quantalan™. Dotted lines show the time of U4-3 administration.

(3) FIG. 3: Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as an indicator of liver function. The results are shown for administration of U4-3 alone (.circle-solid.), U4-3 in combination with Ursofalk™ (.square-solid.) and U4-3 in combination with Quantalan™ (.box-tangle-solidup.). Dotted vertical lines illustrate the time of U4-3 administration.

(4) FIGS. 4a-4b: Total bile acid levels in serum of cynomolgus monkeys. ULN: 13.91 in males, 7.85 in females. The results are shown for male animals in FIG. 4a and for females in FIG. 4b. .circle-solid. represents values obtained for U4-3 alone, represents U4-3 in combination with Ursofalk™ and A represents U4-3 in combination with Quantalan™. Dotted lines illustrate the time of U4-3 administration.

(5) FIGS. 5a-5b: FGF19 in serum of cynomolgus monkeys. The results are shown for male animals in FIG. 5a, for females in FIG. 3b and the combined results are shown in FIG. 5c. Values shown with represents administration of U4-3 alone, .square-solid. represents U4-3 in combination with Ursofalk™ and .circle-solid. represents U4-3 in combination with Quantalan™. Dotted lines illustrate the time of U4-3 administration.

(6) FIG. 6: Human IgG in monkey serum. The results shown were obtained for the combination of male and female animals. Values shown with .circle-solid. represent administration of U4-3 alone, .square-solid. represents U4-3 in combination with Ursofalk™ and .box-tangle-solidup. represents U4-3 in combination with Quantalan™. Dotted lines illustrate the time of U4-3 administration.

(7) FIG. 7: Gene expression of liver Cyp7A at the end of the study. The results are shown for the administration of U4-3 alone (.circle-solid.), U4-3 in combination with Ursofalk™ (.square-solid.), and U4-3 in combination with Quantalan™ (.box-tangle-solidup.), in comparison to the untreated control group (.Math.).

(8) FIG. 8: Ileum FGF19 gene expression at the end of the study. The results are shown for the administration of U4-3 alone (.circle-solid.), U4-3 in combination with Ursofalk™ (.square-solid.), and U4-3 in combination with Quantalan™ (.box-tangle-solidup.), in comparison to the untreated control group (.Math.).

EXAMPLES

Toxicity of FGFR4 Inhibitors in the Presence and Absence of an Agent Capable of Reducing Bile Acid Levels

(9) In order to find ort whether any toxic side effects of FGFR4 inhibition are due to elevated bile acid levels, the effect of co-administering agents which interfere with bile acid levels was examined. The anti-FGFR4 antibody U4-3 was used as inhibitor of FGFR4 activity (Test Item: TI). For reducing bile acid levels, the bile acid sequestrant colestyramine (Quantalan™) was used. Ursodeoxycholic acid (Ursofalk™) was used as a second compound for increasing the transport of bile acids out of the liver. Ursodeoxycholic acid is a hydrophilic bile acid that increases bile acid transporter expression and plasma membrane translocation in the liver, (palliative compounds 1 and 2).

(10) The study design included four cynomolgus monkeys (two male, two female) per group. The animals were assigned to the following groups

(11) TABLE-US-00003 Test item Palliative compound Dose Dose Dose Dose Dose Dose level volume concentration level .sup.a volume concentration Group Treatment (mg/kg) (mL/kg) (mg/mL) (mg/kg/adm) (ml/kg/adm) (mg/mL) 1 TI 100 9.52 10.5 0 2.5 0 2 TI + PC1 100 9.52 10.5 10 2.5 4 3 TI + PC2 100 9.52 10.5 175 2.5 70 .sup.a the daily dose level is given in twice at 8 hours ± 1 hour apart, i.e. a dose level of 20 mg/kg/day for palliative compound 1 and 350 mg/kg/day for palliative compound 2. adm: administration; TI: test item (U4-3); PC1: palliative compound 1 (Quantalan1 ™); PC2: palliative compound 2 (Ursofalk ™)

(12) Administration

(13) Test item (U4-3)

(14) Route: intravenous, slow bolus infusion.

(15) Duration and frequency: three administrations at 7-day interval (i.e. day 0, day 7 and day 14). The first day of dosing was designated as day 0. After the last administration, the animals were kept for a treatment-free period of at east 28 days.

(16) Method: intravenous injection (slow bolus) using a pump system and a microflex infusion set introduced into a vein after local disinfection with an aqueous solution of ethyl alcohol. After injection, the vein was flushed with sterile physiological saline.

(17) Three veins were used in rotation: Right external saphenous vein: site 1. Left external saphenous vein: site 2. Right cephalic vein: site 3.

(18) Volume administered: 9.52 mL/kg/day. Individual dose volumes were calculated using the latest body weight.

(19) Duration of dosing: approximately 15 minutes.

(20) Rationale for choice of route of administration: the intravenous route selected as this is a potential route of administration in humans.

(21) Palliative Compounds and Vehicle

(22) Route: oral.

(23) Frequency: twice daily at 8 hours±1 hour apart.

(24) Duration: each day from day 0 to the last day of treatment-free period, i.e. at least 41 days of administrations.

(25) Method: naso-gastric intubation (animals are trained to this administration rotate five days before the start of treatment).

(26) Fruit or vegetable was given before the daily administration; pelleted diet was given after the daily administration. Palliative compounds were maintained under magnetic stirring from 15 minutes before and during the dosing.

(27) Volume administered: 2.5 mL/kg/adm. Individual dose volumes were calculated using the latest body weight.

(28) Rationale for choice of route of administration: the oral route was selected as this is the route of administration in humans.

(29) Observations

(30) The test animals were examined for clinical signs, liver enzymes, bile acid levels and FGF19. FGF19 serum levels were determined using quantiquine human FGF19 ELISA from RND according to the manufacturer's instructions. RNA expression of cyno liver and ileum samples for Cyp7A1 (liver) and FGF19 (Ileum) were determined using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems/Life Technologies) followed by subsequent quantitative real-time PCR. The results are shown in FIGS. 1-8 and in Table 1 below.

(31) Liver enzyme levels in the serum of cynomolgous monkeys were determined to show that enzyme levels by bilirubin, γ-glutamyltransferase and alkaline phosphatase are not affected by administering the FGFR4 inhibitor with or without bile acid-modifyingagents. (FIG. 1) Elevated levels of liver enzymes (ALAT), bile acids and FGF19 were found in cynomolgus serum after U4-3 administration in line with the suggested biological function of FGFR4 in bile acid level regulation. Some animals also showed minimal bile duct hyperplasia after histopathological examination of the liver. All these increases and changes were abolished by co-administration of Quantalan™ (colestyramine). With Ursofalk™, the effect was less pronounced (FIGS. 2 to 5). Human IgG levels in serum were equal in all treatment groups showing that Quantalan™ and

(32) Ursofalk™ did not work by simply reducing serum antibody levels (FIG. 6) as can be expected by the different organ compartments in which the co-treatments work (FGFR4 in blood accessible organs like the liver whereas Quantalan™ and Ursofalk™ are oral compounds and Quantalan™ is directly excreted without crossing into the blood stream). The elevated liver Cyp7A gene expression at study end shows that the FCFR4 antibody inhibits liver FGFR4 in all treatment groups until the end of the study further supporting that the FGFR4 inhibitor function of the antibody is unchanged by the co-administered compounds (FIG. 7). Co-adminstration of Quantalan™ (colestyramine) with the FGFR4 antibody also prevented the elevated FGF19 expression levels in the ileum compared to FGFR4 antibody alone. This reiterates that bile acids upregulate FGF19 in the gut (FIG. 8).

(33) TABLE-US-00004 TABLE 1 Overview of the changed blood clinical chemistry values in the cyno study (100 mg/kg weekly × 3 + 28 d recovery) Male (100 mg/kg/adm) pretest day 14 day 42 Bile acids (μmol/L) 2.31 42.90 24.16 Alkaline phosphatase (IU/L) 1985 2452 2490 Aspartate aminotransferase (IU/L) 43 158 119 Alanine aminotransferase (IU/L) 37 234 223 Female (100 mg/kg/adm) pretest day 14 day 42 Bile acids (μmol/L) 1.08 69.14 21.77 Alkaline phosphatase (IU/L) 1030 1410 1200 Aspartate aminotransferase (IU/L) 34 192 57 Alanine aminotransferase (IU/L) 35 332 131

(34) Conclusion

(35) Under the defined experimental conditions, three intravenous administrations of U4-3, 7 days apart at the dose level of 100 mg/kg, alone or associated with two different palliative compounds (Ursofalk™ at 20 mg/kg/day or Quantalan™ at 350 mg/kg/day; oral administration twice a day at 8 hours apart) were clinically well tolerated, without clinical signs, local reaction, nor effect on food consumption or body weight. Elevated serum bile acids and liver enzyme levels (aspartate aminotransferase and alanine aminotransferase) were noted in animals receiving the FGFR4 inhibitor alone or together with the palliative compound 1, Ursofalk™.

(36) Treatment with the test item associated with palliative compound 2, Quantalan™, only induced transient aspartate aminotransferase activity. The effect was reversible at the end of the 4-week treatment-free period. The addition of Quantalan™ permitted to maintain bile acid concentration (FIG. 2) and alanine aminotransferase activity within the normal range (FIG. 3) and there were no histopathological changes

(37) The histopathological examination did not reveal any item-related findings.

(38) Administration of Ursofalk™ or Quantalan™ as adjunct treatment had no impact on the systemic exposure to U4-3.

(39) Based on these findings, the test item U4-3 at the dose level of 100 mg/kg was considered to be better tolerated when it was administered in association with the palliative compound 2, Quantalan™.

(40) The study therefore confirmed that side effects of the administration of an FGFR4 inhibitor (U4-3) can be significantly reduced by additionally administering an agent capable of reducing bile acid levels (in particular colestyramine). The combination therapy of U4-3 and colestyramine resulted in no toxicological findings (clinical signs, hematology, blood chemistry and histopathology), whereas these parameters have been found elevated after treatment with U4-3 alone.

(41) The present specification also includes an ASCII text file entitled “WK10053-0004USD1-59300PEP-ST25-replacement-seq-listing-FINAL-10-13-20.txt” that was created on Oct. 13, 2020, and has a file size of 42,527 bytes, the subject matter of which is incorporated by reference in its entirety.