Bisphosphonates and pharmaceutical compositions containing them

113246/2

BISPHOSPHONATES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

Eitan, Pearl, Latzer & Cohen-Zedek

P-64974-IL 113246/2 ·· FIELD OF THE INVENTION:

There are provided novel derivatives of certain bisphosphonates, and especially of compounds of the pamidronate and alendronate type. More particularly, the invention relates to antinoacyl and peptidyl derivatives of such compounds. Furthermore, the invention relates to pharmaceutical compositions which contain as active ingredient an adequate quantity of such novel compounds. The novel pharmaceutical compositions are characterized by improved absorption in the human body after application by the oral route. 1 BACKGROUND OF THE INVENTION:

Calcium-related disorders in general and osteoporosis in particular are a major public health problem in developed countries. Several important pathological conditions are calcium-related and involve irregularities in calcium metabolism: Paget's disease, osteroporosis, hypercalcemia of malignancy, and osteolysis from bone metastases, etc.

Bisphosphonates are a relatively new family of drugs used clinically in various calcium-related disorders including tumor osteolysis, and are undergoing clinical trials for osteoporosis. They are poorly absorbed following oral administration probably due to their high polarity and charge. IV and IM administration is a serious obstacle to their wide-spread use.

BISPHOSPHONATES

Bisphosphonates have been approved for clinical use in

Paget's disease, tumor osteolysis, and hypercalcemia of malignancy and approved in some countries for osteoporosis therapy. Most bisphosphonates are disodium salts of the tetraacids (M.W. approximately 250), and are poorly absorbed from the Gl tract (approximately 1% of 2

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the oral dose is absorbed). Chronic IM or SC administration of bisphosphonates causes irritation and

necrosis, and the oral route has been associated with GI disturbances, resulting in poor patient compliance and side effects. For example, the treatment protocol of pamidronate in tumor osteolyis is 1-day slow and diluted IV infusion to avoid thrombophlebitis, but treatment is repeated if normocalcemia is not attained. Another example is the chronic therapy (years) required in osteoporosis . ABSORPTION BARRIERS:

Clinically, the oral route.is the most common and accepted one for delivering drugs of a low molecular weight, of up to 400-600. However, the low permeability of the intestinal epithelia towards highly polar and charged molecules impedes the effective absorption of many low molecular weight drugs. Many such drugs must be delivered parenterally by frequent injections. This is highly risky without close medical supervision. The problem is particularly acute in cases of drugs used for treatment of various chronic diseases such as cancer and 3 113246/2 age-related diseases, such as osteoporosis, which require prolonged drug treatment.

NEW DRUGS FOR CALCIUM-RELATED DISORDERS

Drugs require a degree of lipophilicity to pass through the GI barrier. In order to increase oral absorption of drugs with low membrane permeability, nonpolar prodrugs are often utilized. Due to the wide variety of esterases present in the target tissue for oral prodrug-

regeneration, esters are the most common prodrugs when GI absorption is considered. Acyloxymethyl esters of bisphosphonic acids were proposed however this did not lead to a useful drug (European Patent EP 0 416 689 A2 date of filing 29.08.90). similarly, Fels et al. proposed pharmaceutical compositions comprising bisphosphonates and sodium lauryl sulfate for increased oral absorption (US Patent 4,980,171, 12.25.90 and IL 89868).

One way to increase membrane permeability of drugs is by utilization of the peptide carrier system (G.L. Amidon, P.J. Sinko, M. Hu, and G.D, Leesman, In L.F. Prescott, and W,S, Nimmo (eds.). 4

Thus, the present invention relates to novel compounds of the general formula H_N ( CHCONH) 2 J , R1 CHCONH- Q -nl R where Q is Y or -(CH„) -c m CH - 7 C n is zero or an integer, i PO~H9 I 3 2 m is 2, 3 or 4, R is a side chain of an amino acid, R1 is a side chain of an amino acid, X is -H or -OH, Y is -H ot -NR2R3, where A designates a 5- or 6-membered heterocyclic ring which contains 1, 2 or 3 nitrogen atoms, zero, 1 or 2 oxygen atoms and which may contain a sulfur atom, which contains up to and including 3 double bonds, where R2 and R3 are independently hydrogen, lower alkyl, lower 5 alkenyl, lower alkoxy, (di )alkylaminoalkyl, alkoxyalkyl and where the ring A may be substituted by one or more t conventional substituents, and to pharmaceutical compositions of improved absorption form the gastrointestinal tract which contain as active ingredient an

A efficient quantity of a compound defined above.

Preferred compounds are of the formula PO3H2 H2W (CHCONH )It CHCONH (0¾) mi-0H ill . ll ^O3H2 / wherein n designates zero or an integer, m designates 2, 3 or 4; and where R1 and R2, which may be identical or different each designates a side chain of an amino acid

The compounds defined above are aminoacyl derivatives when n is zero, and they are peptidyl-bisphosphonates when n is an integer as defined above. A wide range of amino acids can be used, and preferred ones for use in the peptidyl chain according to the invention are: 6 proline plenylalanine alanine lysine arginine

The invention furthermore relates to a method for the production of derivatives defined above, which comprises linking a desired bisphosphonate compound, of the

Pamidronate or Alendronate type, to one or more amino acids .

The invention further relates to pharmaceutical compositions, for oral administration, which contain an effective quantity of a novel derivative defined above.

The compositions according to the present invention are characterized by high absorption from the gastrointestinal tract.

The effective dosage range is of the order of from about 0.01 mg to about 100 mg per patient per day, a preferred range being of the order of 0.2 mg to about 10 mg per day. Compositions according to the invention can be in 7 any acceptable oral dosage form. For some applications, enteric coated forms are preferred.

In the following, the invention is described by way of illustration only with reference to representative examples. It ought to be clearly understood that the novel compounds can contain from one amino acid "elongation" and up to a multi-amino acid-residue peptidyl chain.

The novel compounds are effectively absorbed and after being absorbed, and due to enzymatic action, decomposed to provide the free active.drug. It is preferred to use such peptidyl chains which are effective in balancing the negative charges of the bisphosphonates. It is possible that some of the novel conjugates are effective as such in the human body.

Peptidyl prodrugs of clinically approved bisphosphonates are effectively absorbed following oral administration.

The present prodrug strategy was based on the rationale of neutralizing the negatively charged bisphosphonate molecule by a positively charged amino acid, and or at 8

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the same time making use of the peptide carrier system serving as a transporter for the prodrug.

Following membrane transport, the prodrug is subsequently hydrolyzed by a mucosal cell cytosolic enzyme such as prolidase, prolinase, dipeptidase, aminotripeptidase or possibly other hepatic/plasma enzymes, or is effective as such.

The rationale for the synthesis of peptidylbisphos- phonates is twofold: a) a peptidylbisphosphonate can be recognized by the nonspecific peptide transporter, and b) the free amino groups on the amino acid side chain are expected to neutralize partially or fully the phosphonate negative charges. Amino acids and peptides were linked to geminal-aminoalkylidenebisphosphonates (for example

Pamidronate and Alendronate) by a simple chemical procedure to afford aminoacyl - and peptidylbisphos- phonates. Aminoacyl or peptidyl bisphosphonates thus obtained are reconverted to the parent drug by enzymes or alternatively may be active as such in bone diseases.

Synthesis of a representative example: 9 L-Prolyl-L-phenylalanylpamidronate (Pro-Phe-Pam, see formula). Ν,Ν'-dicyclohexylcarbodiimide (520 mgz 2.52 mMol) was added to a / ' stirred and ice cooled &lt; . ., ./ / —NH—fCHah—i—, o

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—NH—CH Fhbi solution of Boc-L-Pro-L-PheOH (660 mg, 1.82 mMol) and N-hydroxysuccinimide (NH5, 230 mg, (2 mMol) in dichloromethane (DCM, 12 ml). After stirring for 30 min at 0° the solution was further stirred for 48 h at room temperature. The precipitated dicyclohexylurea was filtered washed with DCM (10 ml). The solvent was evaporated, and the residue was treated with pentane (25 ml). The crystalline product was filtered and washed with pentane to give 803 mg N-t-butoxycarbonyl-L-prolyl-L-phenylalanine N-hydroxysuccinimide ester (Boc-Pro-Phe-ONHS). This compound was added to a solution of 3-amino-1-hydroxypropane-1,1-bisphosphonic acid (277 mg, 1.18 mMol) and diisopropylethylamine (0.71 ml, 4.15 mMol) in 1.7 ml isopropyl alcohol and 0.25 ml water. After stirring the solution for 48 h, the solvent was evaporated to afford a syrup. To the residue was added 13 ml water and 0,53 ml acetic acid to bring the pH to about 3. The solution was filtered to remove non phosphorus containing impurities, and the filtrate was evaporated. To the residue was added 13. ml of concentrated (14%) solution of benzathine diacetate in water, causing the formation of a viscous syrup. After 2 h the supernatant liquid was removed by decantation and a new portion of 13 ml benzathine solution was added and the mixture was left overnight. The supernatant was decanted again 10 and the viscous syrup was washed with water (3x5 ml) and ether (2x12 ml), the washing solutions were removed by decantation. The syrup was dissolved in a solution of water-MeOH (1:2, 80 ml) and the solution was passed through a column of Dowex 50 (H* form, 30 ml) and the column was washed with water-MeOH (1:2, 80 ml) till a pH of 4-5 was obtained in the eluted liquid. The solution was evaporated to give a mixture of N-t-butoxycarbonyl-L-prolyl-L-phenylalanylpamidronate and of L-prolyl-L- phenylalanylpamidronate. To this mixture 1.8 ml of trifluoroacetic acid was added to complete the removal of the t-butoxycarbonyl (Boc) protecting group. After 30 min at room temperature the reaction mixture was evaporated to dryness followed by coevaporation with MeOH (5x25 ml). The solid obtained was filtered and washed with MeOH to yield 388 mg (70%) of the final product. NMR: (D20 with Na2CO3, pH=7): 3,P (H decoupled) 17.9 ppm; ‘H: 1.4-2.4 (m, 6K); 3-3.5 (m, 6H); 4.25 (m, 1H); 4.52 (m, 1H); 7.2-7.4 (m, 5H). M.W. Calcd.: 479.36. Found by FAB MS: 479.8.

Biological Experiments

Pro-3H-Phe-MC-Pamidronate and ‘“'C-Pamidronata were administered orally by intragastric intubation to rats weighing 250 g, housed in metabolic cages, in doses of 10 mg. As can be seen in Chart 1a, Pro-Phe-Pam is effectively absorbed following oral administration: 28.4±6.5% and 1.37+0.4% were recovered in the urine and in the tibia after 24 h, in comparison to only 0.3±0.007% and 0.5±0,011% in the same organs following 11

Pamidronate administration. This effective absorption of the prodrug is also evident by the low drug content found in the feces (2.3±0.5%) in comparison to markedly high content of the drug in the feces after Pamidronate administration (91.3+2.8%).

This excellent oral absorption is comparable to the results obtained following IV administration, as reported by Wingen and Schmahl and illustrated in Chart 1b: 19.5% and 0.6% were recovered in the urine and the tibia of rats, respectively.

It is important to note that following the prodrug administration, more of the parent drug, Pamidronate {14C labeled) was found in the bones and in the urine than the prodrug (3H and l4C labeled). On the other hand, significantly higher levels of the amino acid(s) (3H labeled) were detected in the soft tissues, especially in the intestinal wall, than the prodrug (labeled both by 3H and ,4C ) or Pamidronate alone (t4C labeled). This demonstrates that the prodrug was hydrolysed in the body fluids.

Experiments were also carried out with certain compounds according to claim 1, having a heterocylclic ring in the molecule.

Results similar to the ones demonstrated above and in the

Figure were obtained with these.