Background

Glucosamine (GlcN) is a naturally occurring amino-sugar that has been used to treat various ailments including osteoarthritis (OA). Animal studies suggest potent antiinflammatory and disease modifying effects for GlcN. The reported human clinical trials are, however, inconclusive (1 -10), with efficacy reported ranging from negligible to as mush as those reported for NSAIDS. In addition, plasma GlcN concentration following effective doses in rats (~300 mg/kg) (11 ,12,13) are several magnitudes greater than those expected or observed following human doses (1500 mg/day) (13). Similarly, GlcN plasma concentrations of products with beneficial effects in the treatment of OA (14), although variable, are substantially higher than those with no or negligible effects (13).

Various studies suggest limited benefit to the use of natural glucosamine to the following points:

(a) GlcN is not a regulated compound, and the quality of the available products (and also those used in clinical trials) is questionable. It has been reported that most commercially available products contain substantially less GlcN than the label claim (15).

(b) The dosing regimens used are empiric because of scant pharmacologic

information. A dose-effect study using a pharmaceutical grade GlcN formulation is lacking. Indeed, clinical trials have typically concentrated on a 1500 mg/day (label claim, very likely <1500 mg/day). Only animal studies and human trials that have been carried out with relatively high doses or were associated with high plasma GlcN concentrations have reported pharmacological efficacy. GlcN has a low and erratic bioavailability following oral dose, so excessively large doses are needed for therapeutic benefit.

(c) Due to its physical instability, GlcN is often crystallised along with considerable amount of KCI, hence, a typical tablets containing 500 mg GlcN weighs around 1.4 g. The mere size of the available tablets deters patients from taking these pills more than 500 mg t.i.d. to experience the beneficial effects of higher amount in the body.

(d) We hypothesized that pro-drugs of GlcN and other amino-sugars such as butyryl glucosamine will possess relatively high bioavailability that yield high

concentrations, hence, will be effective against OA and rheumatoid arthritis (RA). Therefore, developing pro-drugs that yield high bioavailability of GlcN is beneficial. l Brief Description of Drawings

Figure 1 Peptide-GlcN ester and peptide-GlcN amide derivatives.

Figure 2 The peptide-GlcN ester derivatives are synthesized using solid-phase synthesis on a 2-chlorotrityl chloride resin.

Figure 3 The glycine-valine sequence used for (5-amino-3,4,6-trihydroxyoxan-2- yl)methyl 2-(2-aminoacetamido)-3-methylbutanoate or glycine-valine-COO-GlcN (GV- GlcN)

Figure 4 compares the absorption through everted rat gut of GlcN with GV-GlcN.

Figure 5 tests the stability of the pro-drug of the present invention as it passes through a membrane. Figure 6 shows that the bioavailability of the pro-drug GV-GlcN is substantially higher than natural glucosamine (GlcN).

Figure 7 compares bioavailability in terms of mean plasma concentration.

Figure 8 shows that the pro-drug is superior to natural glucosamine in preventing adjuvant arthritis.

Figure 8 shows that the pro-drug is superior to natural glucosamine in treating or controlling adjuvant arthritis

Summary of Invention

The present invention is a pro-drug of GlcN with the following properties that render the invention superior to GlcN in both bioavailability and therapeutically beneficial, e.g., effective against adjuvant arthritis (AA), osteoarthritis (OA), and/or rheumatoid arthritis (RA):

(a) Physicochemically stable so that it can be manufactured to a tablet size that is easy-to-swallow;

(b) Relatively stable in the gastrointestinal tract. Figure 4 shows that GV-GlcN is stable (greater than 96%) after 60 minutes exposure to the gut, that is, GV-GlcN does not convert into glucosamine within the 60 minutes ;

(c) Efficient in crossing the gut membrane as the intact pro-drug; see Figures 4 and 6.

(d) Efficient conversion to GlcN in the liver, consequently delivering high amounts of GlcN to the systemic circulation. Example 12 shows that the pro-drug (GV-GlcN) converts to glucosamine in about 5 minutes. Figure 7 shows blood concentration.

The compounds described in the present application are esters and amides with a preferred ester having the desired properties as a pro-drug. Compounds described herein are structurally different from those reported in Patent CA 1239636, which are the ether derivatives of GlcN, in which one of the secondary alcohol group of GlcN is conjugated to the peptide by an ether linkage. Design and Synthesis of Peptide-GlcN Derivatives.

To increase gut absorption, we have synthesized pro-drugs that are designed as peptide-GlcN conjugates. This principle has been used earlier for other structurally different compounds (e.g., (16), United States patent numbers 5,831 ,075; 7,371 ,809; 4,548,819;4,957,924).

The present invention is a composition comprising a glucosamine pro-drug as the active agent. As used herein, glucosamine pro-drug refers to Gly-Val-COO-GlcN (GV- GlcN). The composition may also include other ingredients that are commonly used in conventional glucosamine formulations, including but not limited to chondroitin, talcum, lactose, magnesium stearate, binders, fillers, flavors, lubricants, disintegrants, and coatings. The preferred composition is a tablet.

The present invention is also the use of the active agent and/or composition described above for any purpose that provides therapeutic benefit. Examples of therapeutic benefit include but is not limited to the prevention or treatment of osteoarthritis, rheumatoid arthritis, and adjuvant arthritis.

Example 1.

Of several GlcN functional groups, the primary alcohol and the amine were used to make peptide-GlcN ester and peptide-GlcN amide derivatives, respectively (Figure 1 ). Several mono- and di-amino acid derivatives of GlcN, such as, Val-COO-GlcN, Phe- COO-GlcN, Val-CONH-GlcN, Phe-CONH-GlcN, Val-Val-COO-GlcN, Phe-Phe-COO- GlcN, Val-Val-CONH-GlcN, and Phe-Phe-CONH-GlcN, were synthesized.

Example 2.

The peptide-GlcN ester derivatives were synthesized using solid-phase synthesis on a 2-chlorotrityl chloride resin as described before for other compounds (17,18). As depicted in Figure 2, in a typical solid phase synthesis, GlcN may be dissolved in dimethylformamide/triethylamine (DMF/TEA) and added to the pre-swelled resin in dichloromethane (DCM). Coupling between the resin and GlcN is achieved by stirring the mixture overnight at room temperature (19). The resin is then drained to remove all the reagents and solvents followed by washing with DMF and DCM.

In the next step, the Fmoc or Boc protected amino acid were activated by the addition of BOP, HOBt, and NMM in DMF. The activated amino acid or dipeptide was then added to the resin and the mixture was stirred for several hours at room

temperature. The primary alcohol group of GlcN reacts with the activated carboxylate of the amino acid (or dipeptide). After draining and washing, the resin was treated with 20% piperidine to remove Fmoc from the terminal amino group prior to final cleavage. In the case of Boc protected terminal amino group, the Boc removal was achieved during the final cleavage step.

The final cleavage of the product from the resin was achieved by treating the resin with trifluoroacetic acid (TFA) in DCM. The resulting solution was dried under vacuum, followed by washing with cold ether to obtain the final product. The product may be characterized by mass spectrometry. Some compounds were also characterized by reversed -phase HPLC.

Example 3.

The peptide-GlcN amide derivatives were synthesized in solution. Briefly, GlcN was dissolved in DMF/TEA, followed by addition of pre-activated Boc-amino acid or Boc- protected dipeptide as described above for the solid phase synthesis. The resulting mixture was stirred overnight and water was added to precipitate the Boc-protected peptide-GlcN conjugate. The conjugate was then treated with TFA in DCM to remove the Boc-protection, followed by evaporation to dryness. The product was washed with cold ether prior to characterization by mass spectrometry and reversed-phase HPLC.

Example 4.

Chemical Stability. Compounds were transferred into transparent glass vials and placed in 60 ^° C oven for 48 h. Compound with <96% stability are considered unstable.

Example 5.

Stability in the gut. Pro-drugs were incubated for 2 h with homogenized rat stomach or intestine while the pH is kept at 1.0 or 7.4, respectively. Samples were taken at 30 min interval for the analysis of the pro-drug and GlcN. This procedure was used as a prerequisite to further tests described in Examples 6, 7 and 8. A compound with low stability under these conditions was decided to be unsuitable for further testing. Figure 4 shows the comparison of absorption through everted rat gut of GlcN and GV-GlcN. Everted rat gut sacks were immersed in 80 g/mL(GlcN equivalent) of GlcN HCI or GV- GlcN HCI solution. The appearance of each compound inside the sack (serosal) was measured. Figure 5 shows the Average % of ratio of GlcN inside over outside the rat gut sack after 60 min for the ester derivatives of GlcN. Gly-Val-COO-GlcN is GV-GlcN.

These comparisons to glucosamine show that the pro-drugs of the present invention exhibit greater bioavailability. Example 6.

Hepatic Metabolism. Male Sprague-Dawley rats were sacrificed under anesthesia; their livers were excised and stored at -80° C before use. Tissues were homogenized in 10 ml of chilled (4° C) DPBS for about 30 seconds with a tissue homogenizer in an ice bath. Subsequently, the homogenates were centrifuged at 12,500 rpm for 25 min at 4° C to remove cellular debris and the supernatant was used for hydrolysis studies. The compounds were incubated in the presence of this preparation at 37° C. Samples were collected over time and analyzed using HPLC. Percent of the prodrug converted to GlcN was measured.

Example 7.

In vivo absorption through everted rat gut. A mid line incision was made in the anesthetised rat abdomen, the intestine was exposed and five 10 cm segments of the jejunum are cut. In ice cold Krebs Henseleit buffer, the intestinal content are removed and each segment are everted over a glass rod, tied from one side, a polyethylene tube is inserted into the other side and tied. The segments are filled with buffer and incubated in a perfusion apparatus containing oxygenated Krebs Henseleit buffer at 37° C. The test compound was added to the mucosal (outer surface of the everted gut) and serial samples were collected from both sides. The percent of the movement from outside (mucosal) to inside (serosal) was measured and compared (Figure 4).

In addition to assessing the ability to cross the gut wall, the data were used to test the stability of the pro-drug in the presence of viable gut membrane. A lack of presence of GlcN indicated stability and lack of hydrolysis to GlcN. Calculation of mass- balance in both sides of the wall was used to account for unchanged pro-drug, i.e., stability.

Example 8.

Bioavailability in the rat. Prodrugs or GlcN HCI or sulfate were administered to rats in single oral doses equivalent to a fixed amount of GlcN. Plasma GlcN

concentrations were measured using HPLC in serial sample collected over a period of time. The areas under plasma-concentration -time curves (AUC or relative bioavailability) were calculated and compared (Figure 6 and 7).

Figure 6 shows AUC ₍ o. ₄₎ (mg.h/L) of GlcN following administration of 1 0 mg GlcN equivalent of GlcN HCI (GlcN, n=11) and GV-GlcN (GVG, n=7) to rats.

Figure 7 shows the mean plasma concentration of glucosamine after

administration of 110 mg/kg (GlcN equivalent) of GlcN, Gly-Val -COO-GlcN (GV-GlcN), Phe-Phe-COO-GlcN, and Val -Gly-COO-GlcN (VG-GlcN). The area under the concentration curve-time was significantly greater for GV-GlcN (the pro-drug) than the other tested compounds. (n=3-5/group), including natural glucosamine (GlcN).

Example 9. Bioavailability in human.

In a randomized fashion GV-GlcN HCI and glucosamine HCI both at 50 mg glucosamine equivalent were orally administered to a healthy male volunteer. The compounds were dispensed in identical capsules which were administered three days apart. As a measure of relative bioavailability, the total urine output was collected for 8 hours and glucosamine measured therein.

In a human subject, the cumulative amount of glucosamine in 0-8 hour urine

(relative bioavailability) following the administered oral doses of glucosamine and GV- GlcN was 1 .32 and 3.17 mg, respectively, indicating a substantial increase in the bioavailability of glucosamine following oral administration of GV-GlcN as compared with glucosamine.

Example 10. Efficacy against Adjuvant Arthritis.

Under anesthesia, rats were injected with 0.2 ml of 50 mg/ml Mycobacterium butyricum suspended in squalene in the base of the tail (day 0). Their paw thickness was measure daily. Within 12-14 days significant increases in paw thickness were noticed, indicative of the emergence if adjuvant arthritis. Repeated daily doses of drugs were administered either right after the time of Mycobacterium injection (to prevent emergence of arthritis) or after the appearance of arthritis (to treat arthritis). Animals were monitored for their arthritis status during the entire length of the experiment.

Efficacy. As depicted in Figures 8 and 9, GV-GlcN is three fold more potent than GlcN in preventing or controlling the emergence of adjuvant arthritis (AA).

On day zero, Mycobacterium butyricum is injected to all rats along with daily doses of glucosamine, GV-GInN or placebo. The placebo group develops severe AA but not the ones that received either treatment. Upon discontinuation of the treatments, AA emerges (Figure 8). The preventive effect parallels that of treatment effect after emergence of AA since treatment for four days with daily doses of either 90 mg/kg GlcN or 30 mg/kg (GlcN equivalent) GV-GlcN significantly reduce the paw thickness enlarged by AA (Figure 9).

In Figure 8, Preventing effect of GlcN HCI (90 mg glucosamine equivalent) and GV-GlcN (GVG) (30 mg glucosamine equivalent) on the emergence of AA caused by Mycobacterium butyricum. The data suggest i) placebo-treated animals develop arthritis; ii) both GlcN and GV-GlcN prevent the disease; iii) a 3-fold greater potency for GV-GlcN as compared with GlcN (30 mg vs 90 mg GlcN equivalent). Keys: AA, adjuvant arthritis; GVG, GV-GlcN. ( ^* denotes significant change from baseline.)

In Figure 9, Significant (p>0.05) reduced paw thickness following four days of daily doses of GlcN HCI (90 mg glucosamine equivalent) or GV-GlcN (30 mg

glucosamine equivalent). The treatments commenced 14 days following injection of by Mycobacterium butyricum when paw thickness had significantly increased. Equal effectiveness of the two treatments suggests a 3-fold greater potency for GV-GlcN as compared with GlcN (30 mg vs 90 mg GlcN equivalent). Example 1 1.

Screening. Amide and esters synthesized as listed in Example 13were tested for their suitability as GlcN pro-drug. Except for one, all of the tested pro-drugs failed at one or more set criteria explained under Summary of Invention. They possessed either no or little stability in the presence of excised gut or did not convert to GlcN when exposed to the rat liver.

Example 12.

GV-GlcN. The glycine-valine sequence used for (5-amino-3,4,6-trihydroxyoxan-2- yl)methyl 2-(2-aminoacetamido)-3-methylbutanoate or glycine-valine-COO-GlcN (GV- GlcN) (Figure 3) surprisingly exhibited acceptable prod rug characteristics. GV-GlcN is physicochemically stable as measured according to Example 4; >96% stable at 60° C for 48 h); it is relatively stable when incubated in the presence of rat gut as measured according to Example 5; t1/2 of decomposition >16 h)); it is readily converted to GlcN by homogenised liver preparations as tested according to Example 6; Complete hydrolysis to GlcN in <5 min); it crosses everted rat gut as measured according to Example 7; -80% transport.

As depicted in Figure 6, the Average % of ratio of GlcN inside over outside the rat gut sack after 60 min for the ester derivatives of GlcN. Gly-Val-COO-GlcN is GV-GlycN.

As depicted in Figure 6, the mean area under plasma concentration -time curve (AUC, bioavailability) of GlcN in rat plasma following administration of GV-GlcN (110 mg GlcN equivalent) was significantly and substantially greater than that following administration of GlcN HCI (110 mg GlcN equivalent). This result can be extrapolated to GlcN sulfate as well since equal AUC values have been were observed for GlcN following oral administration of its HCI and sulfate salt (23). Example 13

Chemical structures of synthesized, characterized, and tested Pro-drugs. Amino acids are abbreviated as Ala, alanine; Asp, aspartic acid; Gly, glycine; Phe,

phenylalanine; Val, valine; Trp, tryptophan; Tyr, tyrosine.

Phe-COO-GlcN

Val-COO-GlcN

Val-Gly-COO-GLcN

Phe-Phe-P-ALA-COO-GLcN

Example 14

Chemical structures of synthesized, characterized, and tested Pro-drugs. Amino acids are abbreviated as Ala, alanine; Asp, aspartic acid; Gly, glycine: Phe,

phenylalanine; Val, valine; Trp, tryptophan; Tyr, tyrosine.

Peptide-GlcN Amide Pro-dru s

Phe- Phe-CONH-GlcN Val- Val-CONH-GlcN

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