WO2007036802A2

WO2007036802A2 - Sublingual dosage form comrising vitamin d analogue, in particular calcitriol

Info

Publication number: WO2007036802A2
Application number: PCT/IB2006/002853
Authority: WO
Inventors: Moshe Flashner-Barak; Vered Rosenberger; Naomi Moldavski; E. Itzhak Lerner
Original assignee: Teva Pharmaceutical Industries Limited
Priority date: 2005-07-07
Filing date: 2006-07-07
Publication date: 2007-04-05
Also published as: WO2007036802A3; GB0513984D0

Abstract

Disclosed are pharmaceutical dosage forms of vitamin D analogues, such as calcitriol, methods for their preparation and uses.

Description

Dosage Form

The present invention relates generally to dosage forms of vitamin D and its analogues. In particular, the present invention relates to dosage forms for sublingual administration of vitamin D and its analogues.

Background

Vitamin D or cholecalciferol is a secosterol that is the natural precursor of the calcium-regulating hormone calcitriol.

Alfacalcidol (1-α hydroxyvitamin D₃) is a synthetic precursor of the calcium- regulating hormone calcitriol.

A large number of vitamin D analogues having therapeutic activity are known j some of which are described in the references discussed below. One of these, calcitriol, which is variously known as "l(alpha),25-dihydroxycholecalciferol" or "1,25 dihydroxyvitamin D₃", is a vitamin D analogue which is the active metabolite of Vitamin D. Calcitriol is therefore active in the regulation of calcium homeostasis, by facilitating the absorption of calcium from the gastrointestinal tract and mediating its utilization in the body. Calcitriol is the preferred form for administration.

Calcitriol is indicated for the treatment of hypocalcaemia and resultant metabolic bone disease in patients with chronic renal failure, those undergoing dialysis and in patients with hyperparathyroidism [I]. 1, 25 (OH)₂D₃ (calcitriol), facilitates the promotion of calcium transport across the small intestine and is essential for the normal development of bone, mediating these biological actions by binding to VDR (vitamin D receptor), a nuclear transcription factor present in the intestinal epithelial cells that regulates gene expression [2]. Physiological daily production of calcitriol is normally 0.5 to 1.0 meg, but is somewhat higher during periods of increased bone synthesis, such as during growth or pregnancy [I].

In uremic patients, however, in which kidney function is impaired, the hydroxylation of 25-hydroxyvitamin D₃, that typically occurs in the renal mitochondria, and that normally results in the production of 1,25, (OH)₂D₃, is not sufficiently robust. The hypocalcaemia and secondary hyperparathyroidism that results are a major cause of the metabolic bone disease of renal failure. Then, exogenously administered alfacalcidol or calcitriol is indicated to promote intestinal absorption of calcium as well as to effect bone absorption and deposition.

Following oral ingestion, calcitriol is rapidly absorbed from the intestine [I]. Based on a pharmacokinetic study comparing the plasma levels of calcitriol following oral versus bolus intravenous administration, it was shown that the Cmax and AUC levels, after oral administration, were significantly lower than after LV. administration, with a calculated oral bioavailability of about 70%. This was explained to be the result of incomplete absorption, local intestinal degradation and/or hepatic metabolism of enterally absorbed calcitriol (first-pass effect) [3]. Peak serum concentrations are reached within 3-6 hours following oral administration of single doses of 0.25 to 1.0 meg calcitriol. The elimination half-life of calcitriol in serum after single oial administration is about 5-8 hours in normal subjects, while in pediatric patients or patients with nephrotic syndrome, the elimination half-life is increased by at least twofold [I].

In addition to its registered indication, i.e., the treatment of hypocalcaemia and hyperparathyroidism, calcitriol has also been evaluated as a potential therapy, either as monotherapy or in combination with bisphosphonates, in the treatment of osteoporosis [5, 6, 7]. Several studies have demonstrated an additive clinical benefit in osteoporosis therapy provided when calcitriol is given in combination with alendronate. In one study, the combination of alendronate and calcitriol clearly demonstrated clinical superiority, with 69-78% of patients on combination treatment demonstrating bone mass intensity increase as compared to 37-48% of patients receiving alendronate monotherapy [7].

Calcitriol and other VDR ligands are also currently under investigation for their presumed antineoplastic activity in prostate and other cancers [8]. The importance of calcitriol in prostate cancer therapy was initially suggested by epidemiological data that showed that low vitamin D exposure increases the risk of prostate cancer [9]. This was followed by in vitro and in vivo data demonstrating the antitumor effects of calcitriol in a number of pre-clinical prostate tumor model systems. In prostate cancer cell lines, calcitriol was shown to enhance antitumor activity of docetaxel [10], paclitaxel [11] and platinum compounds [12]. The mechanism by which calcitriol inhibits cancer cell proliferation and sensitizes cancer cells to cytotoxic agents remains incompletely understood, although calcitriol has been associated with G0/G1 arrest [13], inhibition of angiogenesis [14, 15], and induction of apoptosis [16], among other mechanisms.

The supraphysiologic concentrations of calcitriol required for anti-tumor effects in preclinical models, however, are not achievable in humans with daily dosing of calcitriol because of the potential for hypercalcaemia and hypercalciuria [4, 17, 18]. The introduction of intermittent dosing, therefore, is being investigated as a means to permit substantial escalation of the requisite calcitriol dose. Thus, Phase II studies are currently underway in which a regimen of three times weekly, high dose (8 meg) calcitriol with dexamethasone, [9] or once weekly high doses of calcitriol (1.5 mcg/kg) with docetaxel [19] are being administered to patients with metastatic androgen-independent prostate cancer (AIPC). Study results indicate that the combinations were well-tolerated, reduction in prostate-specific antigen (PSA) levels was achieved in the majority of patients [4, 9, 19], with favorable time to progression and overall survival rates observed [4, 19]. Additional double-blind randomized studies of combination calcitriol plus cytotoxic agents vs. cytotoxic agent monotherapy are underway. Moreover, preclinical evidence suggests that VDR ligands such as calcitriol, may be applicable to other malignancies, such as breast [20], lung [21], bladder [22] and colon [23].

Calcitriol and other vitamin D analogues are known to have profound immunological effects. The effects of calcitriol and other vitamin D analogues on the immune system are manifold and include suppression of T cell activation, shaping of cytokine secretion patterns, induction of regulatory T cells, modulation of proliferation and interference with apoptosis. Calcitriol and other vitamin D analogues further influence maturation, differentiation and migration of antigen-presenting cells [24], thus making these potential immunomodulatory compounds. For example, the clinical efficacy of calcitriol in the treatment of the autoimmune disorder, multiple sclerosis, is under investigation [25-26]. The common denominator in all these proposed treatments, however, is the absolute need to be able to effectively administer high-dose calcitriol or other vitamin D analogues, without running the risk of inducing hypercalcaemia and hypercalciuria. Although intermittent high dose oral delivery may provide the high serum levels of calcitriol or other vitamin D analogue, this may be impractical in terms of patient compliance. Parenteral administration may also provide high serum levels of calcitriol or other vitamin D analogue, but this may also be impractical and undesirable from a patient perspective.

Sublingual delivery, i.e., systemic delivery of drugs through the membrane of the ventral surface of the tongue and the floor of the mouth, offers substantial advantages as a portal for drug entry to the body [27]. The oral mucosa has a rich blood supply; drugs are transported through the deep lingual or facial vein, internal jugular vein, and braciocephalic vein directly into the systemic circulation [28]. The sublingual mucosa is relatively thin and comprised of nonkeratinized tissue, making it relatively permeable and allowing for rapid absorption and onset of action [29]. Following sublingual administration, the drug gains direct entry to the blood, thereby avoiding the hepatic first-pass effect. Contact with the digestive fluids of the upper gastrointestinal tract is avoided. The rate of drug absorption is not influenced by food or gastric emptying rate [28].

US 6,521,608 (Henner, et al.) discloses the use of vitamin D and its analogues for treating tumours and other hyperproliferative disorders. This document suggests that hypercalcemia may be avoided by pulsed administration of the drug. Oral delivery of a tablet formulation is exemplified.

US 6,034,074 (Rodriguez, et al) discloses the use of a vitamin D compound for prevention of ovarian cancer. The vitamin D compound is administered in an amount capable of increasing apoptosis in non-neoplastic ovarian epithelial cells. This document advocates using large doses of vitamin D on an infrequent basis so as to minimize the adverse calcaemic effects associated with more frequent administration. However, this document does not disclose any specific formulation of the vitamin D compound. US 2005/0009793 (Curd) discloses a method for treating liver disease using an active vitamin D compound that preferably accumulates in the liver. The active vitamin

D compound is preferably administered in a pulsed-dose fashion to avoid hypercalcaemia. This document teaches that oral dosage forms and intravenous dosage forms are preferred.

It has been found that with oral dosage forms in particular, there can be significant variations of bioavailability of the vitamin D compound from one patient to another. This causes difficulties in optimising the high dose required to achieve a therapeutic effect whilst avoiding hypercalcaemic effects.

Whilst for example, US 2005/0009793 mentions a large number of possible delivery methods (oral, nasal, sublingual, vaginal, buccal, rectal, intravenous, intramuscular, intraarterial or topical) for administration of an active vitamin D compound for treating liver disease, this document teaches that oral or intravenous forms are preferred. With regards to sublingual administration, there are several restrictions that can limit this form of administration of a drug. In particular, the available surface area for absorption is relatively small [30]. The delivery systems evaluated for sublingual delivery have been limited to rapidly disintegrating tablets, liquid-filled soft gelatin capsules [27], or liquid solutions or suspensions. These systems are designed to give rapid drug release, leading to high local drug concentrations in the sublingual area. As a result of intense sublingual salivary flow, (saliva pools before swallowing and two major salivary ducts enter this area), the drug concentrations are sustained for a relatively short period of time, probably in the order of only minutes [28]. Additionally, drug characteristics such as taste, irritancy, and allergenicity must be taken into account when positing sublingual administration [28]. Thus, not all drugs are suitable for administration via the sublingual route.

As indicated above, the efficiency of calcitriol and other vitamin D analogues for the treatment of certain diseases requires the achievement of high concentrations of the drug. For example, the use of calcitriol and other vitamin D analogues for the treatment of cancers may be most efficient with the highest blood concentrations of calcitriol. Further, the immunomodulatory effects of calcitriol and other vitamin D analogues are particularly sensitive to the blood concentration of the active agent. Thus, the amount of active agent absorbed can have a profound effect on the immunomodulatory effect of the agent. Variability of blood levels of calcitriol or other vitamin D analogue after any form of dosing could be critical to their activity as immunomodulators in the treatment of auto-immune diseases, such as multiple sclerosis and other similar diseases. In view of this, it would be desirable to provide a dosage form of calcitriol or other vitamin D analogue that results in high serum concentrations of the active agent and that shows decreased patient-to-patient variation in bioavailability, thus maximising the therapeutic effects of the calcitriol or other vitamin D analogue.

We have surprisingly found that if one doses sublingually the high dose calcitriol or other vitamin D analogue in a very small volume in a suitable formulation one can obtain results that are significantly lower in patient-to-patient variability in bioavailability than in other forms of dosing. This improved mode of dosing makes calcitriol and other vitamin D analogues more attractive as candidates for treating, multiple sclerosis and related disorders.

Summary of Invention

One aspect of the present invention provides a sublingual pharmaceutical dosage form comprising a vitamin D analogue and one or more pharmaceutically acceptable solvents, the dosage form having a total volume of between 1 μl to 100 μl.

Another aspect of the present invention is a process for the manufacture of a sublingual dosage form comprising combining the vitamin D analogue with one or more pharmaceutically acceptable solvents.

A further aspect of the present invention is a solution comprising a vitamin D analogue and menthol. This solution can be used to manufacture of a sublingual dosage form

Another aspect of the present invention is the use of a vitamin D analogue for the manufacture of a dosage form for the treatment or prophylaxis of a disease which is susceptible to treatment with a vitamin D analogue, wherein the vitamin D analogue is administered sublingually in a dose of at least 1 μg and wherein the dosage form has a volume of between 1 μl to 100 μl.

A further aspect of this invention is to the method of sublingual administration of doses of calcitriol of at least 1 microgram calcitriol in a volume between 1 microliter and 100 microliter in an appropriate formulation to obtain patient-to-patient variability of bioavailability of at least about 20% less than that obtained with oral dosing.

Another aspect of this invention is to the method of manufacture of formulations of calcitriol wherein the calcitriol is dissolved in a stock solution in menthol. Using menthol as the stock solution imparts stability on the calcitriol by allowing the stock solution to be stored in the solid phase.

Another aspect of the invention is a method of low volume sublingual dosing of a vitamin D analogue at doses greater than 1 micrograms for the treatment or prophylaxis of diseases susceptible to treatment or prophylaxis with a vitamin D analogue.

A further aspect of the invention is a method of treatment or propylaxis of a disease susceptible to treatment or prophylaxis with a vitamin D analogue comprising administering a dosage form as defined herein.

Another aspect of this invention is the method of treatment or prophylaxis of diseases which are susceptible to treatment by vitamin D analogues such as calcitriol. Examples of such diseases include such as autoimmune diseases such as multiple sclerosis, inflammatory bowel diseases (IBD), AIDS, or arthritis, other neurological diseases such as fatigue and cerebral palsy, cancer such as ovarian cancer, breast cancer, prostate cancer, lung cancer or bladder cancer or bone disease such as osteoporosis, Paget's disease, or bone metastases disease by delivering calcitriol in an appropriate high dose of at least 1 microgram by sublingual small volume (1 microliter to 100 microliter) delivery.

The invention further provides the use of a vitamin D analogue for the manufacture of a dosage form for the treatment or prophylaxis of a disease which is susceptible to treatment with a vitamin D analogue, wherein the vitamin D analogue is administered sublingually in a dose of at least 1 μg and wherein the dosage form has a volume of between 1 μl to 100 μl. In embodiments of the invention, the administration results in at least 20%, preferably at least 30% reduction in patient-to-patient variability in the bioavailability of the vitamin D analogue when compared to oral administration. The reduction in patient-to-patient variability may be measured by comparing the average area under the concentration vs. time curve (AUC) for sublingual delivery (e.g. calcitriol sublingual liquid solution in accordance with the present invention) compared to the concentration vs time curve for oral delivery (e.g. using 2 x 0.5 μg Rocaltrol^® soft-gel capsules (Roche) administered orally in a unit dose of 1 μg as a reference)

As used herein, the term "vitamin D analogue" refers to calcitriol and homologues or derivatives of calcitriol and also includes vitamin D metabolites. Such analogues include those discussed in US 2005/0009793. In particular, the vitamin D analogues include those analogues of calcitriol having a modified side chain. Vitamin D analogues useful in the practice of the present invention include, but are not limited to calcitriol, alfacalcidol, doxercalciferol, seocalcitol (EB 1089), calcipotriene, lexacalcitol (KHl 060), ZK 157202, ZK 161422 and ZK 159222, maxacalcitol, paricalcitol, tacalcitol, oxacalcitol, GS1590, CB1393 and GS1500. Preferred vitamin D analogues are calcitriol, alfacalcidol, doxercalciferol, seocalcitol, calcipotriene, lexacalcitol, maxacalcitol, paricalcitol, tacalcitol and oxacalcitol. Particularly preferred vitamin D analogues for use in the present invention are calcitriol, alfacalcidol, doxercalciferol and seocalcitol. Calcitriol is especially preferred.

Description of preferred embodiments

Vitamin D analogues including calcitriol can have many effects on the body and are being investigated to treat many disease states other than calcium metabolism. Many of the treatments of these diseases need a high dose of the active agent to show an effect and in some, especially those whose treatment is mediated by immunomodulatory effect, the exact concentration present in the blood may be critical. The therapeutic effects of the vitamin D analogues may be different depending on the blood concentration obtained, with higher blood concentrations showing a different immunomodulatory effect compared to lower blood levels. In the case of calcitriol, with levels that are too low, there may be no immunomodulatory effect at all. When a high dose of the vitamin D analogue is used, it is desired to avoid the gastrointestinal tract, so as to mitigate the adverse effect on calcium balance. Further, it is desired to have as low a patient-to-patient variability in bioavailability as possible, so as to allow control of the immunomodulatory effects of the vitamin D analogue. We have surprisingly found that sublingual dosing with a low volume dosage form gives enhanced bioavailability with a lower patient-to-patient variability.

In one embodiment of this invention the vitamin D analogue is formulated into a liquid composition suitable for small volume dosing. This small volume dosing is between 1 μl and 100 μl, more preferably between 15 and 50 μl, particularly 20 to 30 μl and most preferably about 25 microliters. In the compositions of the present invention, it is preferred that the total volume of the pharmaceutical formulation is between 1 μl and 100 μl, more preferably between 15 and 50 μl, particularly 20 to 30 μl and most preferably about 25 microliters. It has been found that the volume of 25 microliters provides a particularly good balance between the lowering of the patient-to-patient variability and ease of handling, with a 25 microliter drop being easy to dose. The vitamin D analogue is dissolved in appropriate solvents for pharmaceutical use. The dosage form optionally contains one or more antioxidants in the solution. The solutions may further comprise surfactants and absorption enhancers. Examples of suitable solvents are ethanol, polyethylene glycol 400 (PEG400), mono-, di- and triglycerides, propylene glycol, water with surfactants and mixtures thereof. Examples of antioxidants include butylated hydroxyl toluene, butylated hydroxyl anisole, vitamin E and its derivatives such as tocopherol, PEG-1000 succinate, and vitamin A derivatives. Examples of surfactants are Tween 80 or Tween 20, sodium ducosate, glycerol mono caprylate. Examples of absorption enhancer are oleic acid and menthol. The vitamin D analogue may be present in these solutions in amounts anywhere from several weight percent for very low volume (1-5 μl) doses with very high dose (50 - 200 μg) to 0.001% w/w for doses at the lower end of the high dose range (e.g. ~ 1 μg) and the upper part of the low volume range (e.g. ~ 100 μl). For the more preferred dosing volume (15 to 50 μl) the concentration of the vitamin D analogue will range from about 1.5% to about 0.002%. Most preferably the dosing volume will be about 25 μl and the dose between 2 and 10 μg giving concentrations between 0.04% to 0.008% by weight. A most preferred embodiment has a concentration of the vitamin D analogue of about 0.02% w/w. In a particularly preferred embodiment, the dosage form has a total volume of about 25 μl and comprises about 5 μg vitamin D analogue.

Embodiments of this aspect of the invention may use solvents such as triglycerides (e.g. Miglyol series of solvents) as single solvents, may use mixtures of solvents such as propylene glycol and ethanol at about 50% each by weight more preferably about 65% propylene glycol and 35% ethanol and most preferably about 62% propylene glycol, about 32% ethanol and about 5% surfactant such as Tween 80. Further embodiments of this aspect of the invention may use water as the solvent with 15% surfactants such as a mixture of Gelucire 44/14 and sodium ducosate (95:5). A further preferred embodiment of this invention may use a mixture of ethanol and polyethylene glycol 400 along with Tween 80 as a surfactant and oleic acid as an absorption enhancer. One most preferred embodiment of this aspect comprises about 50% ethanol, about 6-10% PEG400, about 30% Tween 80 and about 10% oleic acid.

Another aspect of this invention is the use of menthol for processing the vitamin

D analogue in forming the low volume sublingual dosage forms. Menthol has a dual usage in this context. Menthol is known to have absorption enhancing properties so that its presence in the sublingual formulations will aid in absorbing the vitamin D analogue from a small volume and small surface area of the sublingual membrane. The enhanced absorption should theoretically aid in lowering the variability of said absorption. Equally importantly, menthol has advantages as a solvent over other candidate organic solvents in that it solidifies at room temperature allowing a stock solution of the vitamin D analogue active agent to be formed and stored as a solid. Storing the active agent as a solid solution enhances its stability compared with a liquid solution, thereby making it possible to make many sub lots of the sublingual dosage form from one conveniently made stock solution of the vitamin D analogue in menthol. Since the dose of even "high dose" vitamin D analogue such as calcitriol is quite low (on the order of several to 10's of micrograms) it is convenient to work through diluting stock solutions of the drug and not to weigh out miniscule amounts of the drug for each batch. Some preferred embodiments of formulations of this aspect of the invention are described in Example 2 with the most preferred embodiment of this aspect and the previous aspect of the invention described in Example 1. The most preferred embodiment, as described in example 1, was tested in a pharmacokinetic trial in healthy volunteers and shown to have patient-to-patient variability in bioavailability of about 39% less than the reference oral delivery while showing enhanced bioavailability. Whilst the examples show the preferred embodiment of the invention using calcitriol as the vitamin D analogue active agent, it will be appreciated that the compositions, uses, methods and processes of the present invention can be applied successfully to other vitamin D analogues, such as alfacalcidol, doxercalciferol, seocalcitol (EB 1089), calcipotriene, lexacalcitol (KHl 060), ZK 157202, ZK 161422 and ZK 159222, maxacalcitol, paricalcitol, tacalcitol, oxacalcitol, GS1590, CB1393, CB1093 and GS1500. Preferably, the compositions, uses, methods and processes of the present invention may be carried out using calcitriol, alfacalcidol, doxercalciferol, seocalcitol, calcipotriene, lexacalcitol, maxacalcitol, paricalcitol, tacalcitol and oxacalcitol as the vitamin D analogues. Particularly preferred vitamin D analogues for the compositions, uses, methods and processes of the present invention are calcitriol, alfacalcidol, doxercalciferol and seocalcitol, with calcitriol being especially preferred.

Another aspect of this invention is the method of treatment of diseases which are¹ susceptible to treatment by vitamin D analogues such as calcitriol.. Such diseases include autoimmune diseases such as multiple sclerosis, inflammatory bowel disorders (IBD) including Crohn's disease and colitis, AIDS, or arthritis; other neurological diseases such as fatigue and cerebral palsy, proliferative diseases such as cancer — particularly ovarian cancer, breast cancer, prostate cancer, lung cancer or bladder cancer; or bone diseases such as osteoporosis, Paget's disease, and bone metastases disease. These diseases all need high doses of the vitamin D analogue and all of these diseases, especially the autoimmune diseases, will have their treatment efficacy enhanced by a lowering of patient-to-patient variability thereby allowing better control of drug delivery and design of dosing regimens. Use of the embodiments described herein, especially the preferred embodiments described herein, in doses of 0.014 μg/kg to 2.8 μg /kg of the vitamin D analogue such as calcitriol, more preferably 0.04 μg/kg to 1.4 μg/kg, and most preferably at about 0.07 μg/kg should be most beneficial to developing new treatment modalities for these diseases.

Embodiments of the present invention are further illustrated by the following examples and figures. Figure 1 shows the normalized (to 1 μg) average data for sublingual calcitriol vs. oral calcitriol.

Figure 2 shows the average data for sublingual calcitriol vs. oral calcitriol

Example 1

Sublingual solution manufacture

Menthol, 12 grams, was melted at 5O⁰C and purged with a flow of nitrogen for one hour. The antioxidants butylated hydroxytoluene (267 mg) and butylated hydroxyanisole (267 mg) were added to the menthol melt. The menthol melt was stirred under nitrogen until all the antioxidants have dissolved. Calcitriol (267 mg) was added to the melt which was stirred under a nitrogen atmosphere until all had dissolved. The vessel was tightly closed. The menthol solution solidifies in the vessel on cooling to RT. Store the vessel at -20C.

In a separate vessel ethanol (95%) was purged with nitrogen for two hours. Into a tared vessel 15 grams oleic acid, 45 grams TWEEN 80 and 10.8 grams polyethyleneglycol 400 were weighed. These ingredients were stirred at 40 degrees under a nitrogen atmosphere until they all dissolved. The nitrogen purged ethanol was added (73.2 grams) and stirred to form a solution.

The solid solution of calcitriol in menthol was melted and 1.44 grams were added to the ethanol solution of oleic acid, Tween and PEG. The obtained mixture was stirred under a nitrogen atmosphere for several minutes and cooled to room temperature. This solution was dispensed into dropper bottles. A 25 μl drop of the solution contains 5 μg of calcitriol. The formulation of the final dosage form is shown in Table 1.

Table 1.

% w/w

Calcitriol 0.021

Menthol 0.928

BHA 0.021 BHT 0.021

Oleic acid 10.314

Tween 80 30.941

PEG 400 7.426 Ethanol 95% 50.330

The formed sub lot of the final dosage form of the calcitriol contains more than 5000 doses of 25μl each. The solid solution of calcitriol may be used about 8 times to form more sub lots of the final dosage solution for more than 40,000 doses. It is stable in storage at -20 degrees and for at least 10 days at 4 degrees.

The low volume dose of calcitriol thus formed was tested in a pharmacokinetic study against oral calcitriol.

Pharmacokinetic study

Title: A Single-Dose, Two- Way Crossover Comparative Bioavailability Study of Calcitriol, Between a Liquid Test Formulation of Calcitriol (sublingual solution of Example 1) (5 meg calcitriol) Administered Sublingually, and the Reference Rocaltrol^® (2 x 0.5 meg calcitriol; Roche) Soft-Gel Capsules, Administered Orally, in Healthy Male Volunteers

Overall Study Design and Plan

This was a randomized, open label, two-way, two period, comparative crossover study.

The subjects received their first treatment assignment at the first study period, and were crossed-over at the second study period to the alternative treatment arm based upon their unique treatment order as determined by the computer-generated randomization scheme, for a total of 2 study periods, with a wash-out period of at least 14 days between study sessions.

The treatments were administered to the subjects in a fasted state, as follows:

Administration 1 (A): 1 x 5 meg Calcitriol Liquid Solution (solution from Example 1), administered sublingually in a unit dose of 5 meg, as 25 microliters; Test

Administration 2 (B): 2 x 0.5 meg Rocaltrol^® soft-gel capsules (Roche), administered orally in a unit dose of 1 meg; Reference

Drugs were administered after fasting for at least 10 hours.

The 5.0 meg (25 microliter) drop of calcitriol test solution made in accordance with Example 1 was administered to the first group of subjects to the sublingual area without water. The soft-gel capsules was administered simultaneously to the second group of subjects and swallowed whole with 240 ml of water at room temperature.

For each of the study periods, 15 serial blood samples were collected per subject up to 24 hours following study drug administration, for measurement of calcitriol levels. Pharmacokinetic Blood Sampling

Blood samples (6 ml) for determination of calcitriol plasma concentrations were taken and transferred into vacutainer tubes containing K-EDTA according to the following time points:

"0" hour (pre-dosing), 5, 15 and 30 minutes, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 12.0, 16.0 and 24.0 hours post-dose (in-house), for a total of 15 blood samples per study period.

During the sample collection, the blood was maintained on ice to ensure cooled temperatures the whole time. Following centrifugation, the 6 ml whole blood yielded about 3 ml plasma, to allow for the preparation of three (3) single triplicates of 1 ml plasma each. The process is described below.

Samples were centrifuged within 15 minutes after collection at 150Og for 15 minutes at 4⁰C, and the plasma separated. The plasma was placed into three small (2 ml volume) screw-capped, non-tapered, polypropylene transfer tubes, with at least 1 ml plasma per tube.

The plasma was frozen to a temperature of at least -2O⁰C at the clinical facility and shipped frozen to the analytical site

Analytical Methodology

The analysis of blood samples for calcitriol was conducted via a validated RIA (radioimmunoassay) method, with an analytical range of 3-180 pg/ml.

Results of Pharmacokinetic Study of Sublingual Calcitriol vs. Oral Calcitriol

The results of the calculated pharmacokinetic properties are given in Table 2. All of the pharmacokinetic (PK) calculations were carried out on the corrected data, i.e. the calcitriol concentration at time zero for each volunteer at each session subtracted from each data point. This was done to remove the natural background of calcitriol in each patient. The sublingual calcitriol showed an average area under the concentration vs. time curve (AUC) of 395 (h*pg/ml) per microgram calcitriol compared to 346.7 (h*pg/ml) per microgram for the oral delivery (ratio = 1.14). Therefore we can say that there is an improvement in the bioavailability when delivering sublingually. This improvement is even greater when the ratio of AUC per microgram is calculated on an individual basis and then averaged. The average of the ratio is 1.44 (7.215/5). The Cmax for sublingual calcitriol is 39.6 pg/ml per microgram compared to 37.5 for the oral calcitriol (ratio = 1.06) while the average of the ratios on a per individual basis is 1.17 (5.865/5). The rise in Cmax is therefore proportionally smaller than the rise in bioavailability, but not greatly so.

With regards to patient-to-patient variability in the bioavailability, the data in Table 2 shows that there is less patient-to-patient variability in this parameter when the calcitriol is administered sublingually compared with oral administration. In particular, the %CV ("%CV" at the end of each column in Table 2 represents the patient-to-patient, variability for the particular pharmacokinetic parameter in that column) in the AUC column, which represents the patient-to-patient variability in the bioavailability, shows a value of 42.82% for the reference oral administration, and a significantly lower value of 26.35% for the test sublingual administration. This represents a percentage improvement in the patient-to-patient variability in the bioavailability of about 38% [(42.82-26.35)/42.82].

The average Tmax for the sublingual formulation was 2.3 hours and for the oral 3.4 hours. The sublingual administration delivers more of the drug at shorter time periods as expected for absorption through the sublingual membrane. The calculated cumulative percent of the normalized AUC was 3.7% and 12.3% for one and two hours for the sublingual delivery and 1.5% and 8.8% for one and two hours for the oral delivery.

The variability in the bioavailability as shown by AUC is greatly diminished

(by almost 40%) in the test sublingual formulation compared to the oral reference. This may be attributed to both an excellent micelle forming solution and the very small volume (25μl) of dose administration.

The concentration vs. time data for all the plasma samples are presented in Tables 3-6. Graphs of the normalized average data and of the average data are shown in Figures 1 and 2 respectively.

Table 3. Data for Calcitriol (pg/ml) Sublingual 5μg Dose

Table 4. Sublingual Calcitriol (pg/ml) - Corrected for Background Level

Table 5. Data for Calcitriol (pg/ml) Oral lμg(2x 0.5 μg) Dose

Table 6. Oral Calcitriol /ml - Corrected for Back round Level

Example 2 —further formulations

Using a method similar to that described in Example 1 (i.e. making a stock solution of the calcitriol in menthol) and using the appropriate materials and amounts, the following formulations were produced . All these formulations are stable solutions and can be used for low volume (e.g. ~25 μl) sublingual delivery.

Table 7.

Table 8.

Table 9.

Table 10.

References

I. Physician's Desk Reference - Rocaltrol® (Roche), 58^th Edition, 2004, Montvale, NJ, pages 2914-2916 2. Malloy PJ et al. Hereditary vitamin D resistant rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular responsiveness. J. Clin. Invest., 1997; 99(2):97-304 3. Brandi E et al. Pharmacokinetics of 1,25(OH)₂D₃ and Ia(OH)D₃ in normal and uraemic men. Nephrol Dial Transplant, 2002; 17:829-842 4. Henner WD and Beer TM. A new formulation of calcitriol (DN-IOl) for high- dose pulse administration in prostate cancer therapy. Reviews in Urology, 2003;5(Suppl. 3):S38-S44 5. Nuti R et al. Effect of treatment with calcitriol combined with low-dosage alendronate in involutional osteoporosis. Clin Drug Invest, 2000: 19(l):55-60 6. Malvolta N et al. Calcitriol and alendronate combination treatment in menopausal women with low bone mass. Int J Tissue React, 1999;21 (2):51 -59 7. Frediani B et al. Effects of combined treatment with calcitriol plus alendronate on bone mass and bone turnover in postmenopausal osteoporosis: two years of continuous treatment. Clin Drug Invest, 1998:15(3):235-244 8. van den Bernd GJ et al. Anti-tumor effects of 1,25 -dihydroxyvitamin D₃ and vitamin D analogs. Curr Pharm Des. 2000:6:717-732

9. Trump D et al. High-dose calcitriol (1,25(OH)2 Vitamin D3) + Dexamethasone in androgen independent prostate cancer (AIPC). Abstract #1327. ASCO (American Society of Clinical Oncology), 2000; Genitourinary Cancer 10. Beer TM et al. Weekly high-dose calcitriol and docetaxel in advanced prostate cancer. Semin Oncol, 2001;28:49-55

I I. Hershberger PA et al. Calcitriol (1,25-dihydroxycholecalciferol) enhances paclitaxel antitumor activity in vitro and in vivo and accelerates paclitaxel-induced apoptosis. Clin Cancer Res, 2001;7:1043-1051 12. Moffat KA et al. 1 -alpha, 25 dihydroxyvitamin D3 and platinum drugs act synergistically to inhibit the growth of prostate cancer cell lines. Clin Cancer Res, 1999;5:695-703

13. Campbell MJ and Koeffler HP. Towards therapeutic intervention of cancer by vitamin D compounds. J. Natl Cancer Inst, 1997;89:182-185 14. Majewski S et al. Inhibition of tumor cell-induced angiogenesis by retinoids, 1 , 25- dihydroxyvitamin D3 and their combination. Cancer Lett, 1993;75:35-39

15. Mantell DJ et al. 1, alpha, 25-dihydroxyvitamin D3 inhibits angiogenesis in vitro and in vivo. Circ. Res, 2000;87:214-220 16. Park WH et al. Induction of apoptosis by vitamin D3 analogue EB 1089 in NCI- H929 myeloma cells via activation of caspase e and p38 MAP kinase. Br J Haematol, 2000; 109:576-583 17. Osborn JL et al. Phase II trial of oral 1,25 dihydroxyvitamin D (calcitriol) in hormone refractory prostate cancer. Urol Oncol, 1995; 1:195-198 18. Gross C et al. Treatment of early recurrent prostate cancer with 1, 25- dihydroxyvitamin D3 (calcitriol). J Urolo, 1998;159:2035-2039

19. Beer TM et al. Weekly high-dose calcitriol and docetaxel in metastatic androgen- independent prostate cancer. J Clin Oncol, 2003;21 : 123-128

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21. Higashimoto Y et al. 1 alpha,25-dihydroxyvitamin D3 and all-trans-retinoic acid inhibit the growth of a lung cancer cell line. Anticancer Res, 1996;16:2653-2659

22. Konety BR et al. Effects of vitamin D(calcitriol) on transitional cell carcinoma of the bladder in vitro and in vivo. J. Urol, 2001;165:253-258 23. Cross HS et al. Antiproliferative effect of 1, 25-dihydroxyvitamin D3 and its analogues on human colon adenocarcinoma cells (CaCo-2): influence of extracellular calcium. Biochem Biophys Res Comm, 1991; 179:57-62 24. May E et al. Immunoregulation through 1,25 -dihydroxyvitamin D3 and its analogs. Curr Drug Targets. Inflamm-Allergy, 2004;3(4):377-393 25. United States Patent 5,716,946 DeLuca , et al granted February 10, 1998

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Claims

1. A sublingual pharmaceutical dosage form comprising a vitamin D analogue and one or more pharmaceutically acceptable solvents, the dosage form having a total volume of between 1 μl to 100 μl.

2. A dosage form according to Claim 1 in the form of a liquid.

3. A dosage form according to Claim 1 or Claim 2 wherein the total volume is between 15 μl to 50 μl.

4. A dosage form according to any preceding claim wherein the total volume is between 20 μl to 30 μl.

5. A dosage form according to any preceding claim wherein the total volume is about 25 μl.

6. A dosage form according to any preceding claim further comprising one or more pharmaceutically acceptable excipients selected from antioxidants, surfactants, and absorption enhancers.

7. A dosage form according to any preceding claim wherein the vitamin D analogue is calcitriol, homologues or derivatives of calcitriol, or a vitamin D metabolite.

8. A dosage form according to any preceding claim wherein the vitamin D analogue is an analogue of calcitriol having a modified side chain.

9. A dosage form according to any preceding claim wherein the vitamin D analogue is selected from calcitriol, alfacalcidol, doxercalciferol, seocalcitol (EB 1089), calcipotriene, lexacalcitol (KH1060), ZK 157202, ZK 161422 and ZK 159222, maxacalcitol, paricalcitol, tacalcitol, oxacalcitol, GS 1590, CB 1393, CB 1093 and GS 1500.

10. A dosage form according to any preceding claim wherein the vitamin D analogue is selected from calcitriol, alfacalcidol, doxercalciferol, seocalcitol, calcipotriene, lexacalcitol, maxacalcitol, paricalcitol, tacalcitol and oxacalcitol.

11. A dosage form according to any preceding claim wherein the vitamin D analogue is selected from calcitriol, alfacalcidol, doxercalciferol and seocalcitol.

12. A dosage form according to any preceding claim wherein the vitamin D analogue is calcitriol.

13. A dosage form according to any preceding claim wherein the vitamin D analogue is present in an amount of 1 μg or more.

14. A dosage form according to any preceding claim wherein the vitamin D analogue is present in an amount of from 1 μg to 500 μg.

15. A dosage form according to any preceding claim wherein the vitamin D analogue is present in an amount of from 1 μg to 200 μg.

16. A dosage form according to any preceding claim wherein the vitamin D analogue is present in a concentration of from 0.001 wt% to 10 wt%.

17. A dosage form according to any preceding claim wherein the vitamin D analogue is present in a concentration of from 0.005 wt% to 1 wt%.

18. A dosage form according to any preceding claim wherein the vitamin D analogue is present in a concentration of from 0.005 wt% to 0.05 wt%.

19. A dosage form according to any preceding claim wherein the vitamin D analogue is present in a concentration of about 0.02 wt%.

20. A dosage form according to any preceding claim wherein the pharmaceutically acceptable solvent comprises a pharmaceutically acceptable solvent selected from ethanol, polyethylene glycol 400, mono-, di- and triglycerides, propylene glycol, water and mixtures thereof.

21. A dosage form according to any preceding claim wherein the pharmaceutically acceptable solvent is menthol.

22. A dosage form according to any preceding claim comprising calcitriol, menthol and optionally one or more excipients selected from antioxidants, pharmaceutically acceptable solvents, surfactants and absorption enhancers.

23. A process for the manufacture of a dosage form as defined in any preceding claim comprising combining the vitamin D analogue with one or more pharmaceutically acceptable solvents.

24. A process according to Claim 23 wherein the pharmaceutically acceptable solvent comprises menthol.

25. A process according to Claim 24 comprising combining the vitamin D analogue with melted menthol.

26. A process according to Claim 25 wherein the melted menthol is combined with one or more antioxidants to form a mixture which solidifies on cooling.

27. A process according to Claim 26 wherein at least one further pharmaceutically acceptable solvent is combined with the melted mixture.

28. A process according to any of Claims 23 to 27 wherein the pharmaceutically acceptable solvent further comprises one or more of a pharmaceutically acceptable excipient selected from an absorption enhancer and a surfactant.

29. A process according to any of Claims 23 to 28 wherein the vitamin D analogue is calcitriol.

30. A solution comprising a vitamin D analogue and menthol.

31. A solution according to Claim 30 in solid form.

32. A solution according to Claim 30 or Claim 31 wherein the vitamin D analogue is calcitriol.

33. Use of a solution according to any of Claims 30 or 32 for preparing a sublingual pharmaceutical dosage form.

34. Use according to Claim 33 wherein the sublingual pharmaceutical dosage form is as defined in any of Claims 1 to 22.

35. Use of a vitamin D analogue for the manufacture of a dosage form for the treatment or prophylaxis of a disease which is susceptible to treatment with a vitamin D analogue, wherein the vitamin D analogue is administered sublingually in a dose of at least 1 μg and wherein the dosage form has a volume of between 1 μl to 100 μl.

36. Use according to Claim 35 wherein the administration results in at least 20% reduction in patient-to-patient variability in the bioavailability of the vitamin D analogue when compared to oral administration.

37. Use according to Claim 36 wherein the administration results in at least 30% reduction in patient-to-patient variability in the bioavailability of the vitamin D analogue when compared to oral administration.

38. Use according to any of Claims 35 to 37 wherein the disease is selected from an autoimmune diseases, neurological diseases, proliferative disorders, and bone diseases.

39. Use according to Claim 38 wherein the autoimmune disease is multiple sclerosis, inflammatory bowel disease, AIDS, or arthritis.

40. Use according to Claim 38 wherein the neurological disease is fatigue or cerebral palsy.

41. Use according to Claim 38 wherein proliferative disorder is a cancer.

42. Use according to Claim 41 wherein the cancer is selected from ovarian cancer, breast cancer, prostate cancer, lung cancer or bladder cancer.

43. Use according to Claim 38 wherein the bone disease is osteoporosis, Paget's disease, or bone metastases disease.

44. A method of low volume sublingual dosing of a vitamin D analogue at doses greater than 1 micrograms for the treatment or prophylaxis of diseases susceptible to treatment or prophylaxis with a vitamin D analogue.

45. A method according to Claim 44 wherein the dose is administered in a volume of between 1 μl and 100 μl.

46. A method according to Claim 45 wherein the dose is administered in a volume of between 15 μl and 50 μl.

47. A method according to Claim 46 wherein the dose is administered in a volume of between 20 μl and 30 μl.

48. A method according to Claim 47 wherein the dose is administered in a volume of about 25 μl.

49. A method of treatment or propylaxis of a disease susceptible to treatment or prophylaxis with a vitamin D analogue comprising administering a dosage form as defined in any of Claims 1 to 22.