EP0540631A1 - Trans-mucosal drug preparations and trans-mucosal administration - Google Patents

Abstract

The invention relates to a pharmaceutical preparation which has a form and a composition suitable for administration by the transmucosal route, in particular the nasal route, and which comprises at least one medicament based on (poly) peptides or proteins or a fragment or analogue of this drug, as an active agent, as well as dimethyl-beta-cyclodextrin as an absorption promoting agent. A method which allows the administration by transmucosal route of a drug based on (poly) peptides or proteins or a fragment or analog thereof, consists in administering a pharmaceutical preparation which has a form and a composition suitable for allowing its administration by the route transmucosal, especially nasal, and which contains dimethyl-beta-cyclodextrin as an agent promoting absorption. The polypeptide preferably consists of insulin or calcitonin.

Description

Title: Trans-mucosal drug preparations and trans-mucosal administration

This invention relates to a pharmaceutical preparation of a form and composition suitable for trans-mucosal,

particularly nasal administration, which preparation comprises at least one (poly)peptide or protein drug, or a fragment or analogue thereof, as an active agent. The invention further relates to a method of trans-mucosally, particularly nasally, administering a (poly)peptide or protein drug, or a fragment or analogue thereof, comprising administering a pharmaceutical preparation of a form and composition suitable for transmucosal, particularly nasal administration.

Insulin is a polypeptide hormone of 51 amino acids. It is produced in the pancreas and functions as a physiological regulator of the carbohydrate metabolism in the body. For many years now insulin has been therapeutically used in patients with diabetes mellitus, to lower increased blood-sugar levels in these patients. However, oral administration is not possible in the case of drugs with a peptide structure, since they are broken down in the gastro-intestinal tract. Therefore these substances must be administered by injection

(subcutaneously, intramuscularly, intravenously). These invasive methods of administration are satisfactory in terms of therapeutic effectiveness, but the daily administration causes the patients considerable inconvenience: injections are experienced as painful and traumatic, are irreversible, and the patient must learn the injection technique.

Alternative, non-invasive routes of administration offer new perspectives for the therapy with (poly)peptides, which includes insulin. In particular, the nasal route of

administration offers a number of advantages: the nasal cavity is easily accessible for drug administration, the nasal epithelial tissue has a rich vasculature, the nasal route prevents the first-pass effect through the liver and is eminently suitable for self-medication.

However, intranasal administration of insulin leads to poor bioavailability, since insulin is a high-molecular and hydrophilic substance and therefore is hardly capable of passing the lipophilic epithelial barriers. However, the nasal bioavailability can be improved considerably by using socalled absorption enhancing adjuvants. In the literature many absorption enhancers have been described for the nasal administration of insulin, including ionic and non-ionic surfactants such as bile salts (Gordon et al., Proc. Natl. Acad. Sci. U.S.A. 82 (1985) 7419-7421; EP-A-0 111 841) and polyoxyethylene alcohol ethers (Hirai et al., Int. J. Pharm. 9 (1981) 165-172; British Patent No. 1 527 605), fatty acids and phospholipids (Mishima et al., J. Pharmacobio-Dyn. 10 (1987) 624-631; Ilium et al., Int. J. Pharm. 57 (1989) 49-54), chelating agents such as EDTA (U.S. Patent No. 4 476 116), and fusidic acid derivatives such as STDHF (Longenecker et al., J. Pharm. Sci 76 (1987) 351-355; Deurloo et al., Pharm. Res. 6 (1989) 853-856; U.S. Patent No. 4 548 922).

These substances all lead to an increased, but not to full bioavailability of nasally administered insulin.

Moreover, the reproducibility of the resulting insulin

absorption profiles in the blood leaves to be desired, many of these absorption enhancers are harmful to the nasal epithelial membranes (Wheatley et al., J. Controlled Rel. 8 (1988)

167-177; Ennis et al., Pharm Res. 7 (1990) 468-475), and various of these substances inhibit the natural movement of the cilia in the nose (Hermens et al., Pharm Res. 7 (1990)

144-146). The epithelial tissue of the nose is covered with a layer of mucus which is transported to the pharynx by the ciliary movement, there to be swallowed and removed via the gastro-intestinal tract. This process, called mucociliary clearance, is an important clearing mechanism of the nose to protect the body against inhaled harmful particles (dust, allergens, bacteria, viruses, etc.). Inhibition of this mechanism may cause serious infections of the airways.

Therefore it is a requisite that drugs and adjuvants in nasal preparations for subchronic or chronic use have no or only a limited influence on the ciliary movement in the nose. There are indications that cyclodextrins may have an absorption enhancing effect on nasally administered

polypeptides such as insulin (EP-A-0 094 157; EP-A-0 308 181). Thus, EP-A-0 094 157 specifies that α-cyclodextrin (α-CD), β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) can improve the absorption of intranasally administered porcine insulin. Those studies were performed in rats. That patent application does not specify any data on the attained insulin concentrations in the blood nor on the bioavailability, but the absorption enhancing effects of α-CD, β-CD and γ-CD are based on measured decreases in the plasma glucose levels (hypoglycemic

responses). The effect of α-CD proved dependent on the α-CD concentration used in the insulin formulation: maximum effects were achieved 2 h after administration and were 60%, 47% and 25% of the initial plasma glucose levels for concentrations of 3%, 5% and 10% α-CD, respectively, in the nasal insulin preparations. The influence of β-CD and γ-CD on the nasal insulin absorption was merely studied in concentrations of 10%: for β-CD a maximum effect of 49% of the initial glucose levels was achieved 2 h after administration, and for γ-CD a maximum effect of 74% of the initial glucose concentrations was found at the time 4 h after intranasal insulin

administration. From the above-mentioned results it can be concluded that of the three cyclodextrins investigated α-CD is the most potent enhancer for nasally administered insulin and γ-CD is the least potent.

EP-A-0 308 181 reports that α-CD, β-CD and γ-CD can improve the nasal absorption of insulin. There, use was made of human insulin and of rabbits as experimental animal model. That patent application, too, does not contain any data on plasma insulin levels or bioavailability, but merely reports data on the influence of the insulin formulations used on the glucose concentrations in the blood. As regards the

hypoglycemic responses an index is used which compares the effect of the nasal insulin preparations with that of a subcutaneous injection preparation for insulin. On the basis of the indices reported in that patent application (Index 42, 44 and 26 for α-CD, β-CD and γ-CD, respectively) it can be concluded that α-CD and β-CD have an equally strong enhancing effect on the nasal absorption of insulin in rabbits, and that the potency of γ-CD is considerably smaller than that of α-CD and β-CD.

The invention provides a pharmaceutical preparation of a form and composition suitable for trans-mucosal, particularly nasal, administration, which preparation comprises at least one (poly)peptide or protein drug, or a fragment or analogue thereof, as an active agent, as well as dimethyl-β-cyclodextrin as an absorption enhancing agent.

The invention further provides a method of transmucosally, particularly nasally, administering a (poly) peptide or protein drug, or a fragment or analogue thereof, comprising administering a pharmaceutical preparation of a form and composition suitable for nasal administration, which

preparation comprises dimethyl-β-cyclodextrin as an absorption enhancing agent.

The present invention is based on the observation that the cyclodextrin derivative dimethyl-β-cyclodextrin (DMβCD) has a much stronger enhancing effect on the absorption of

intranasally administered human insulin. In comparison with α-CD, β-CD, γ-CD and hydroxypropyl-β-cyclodextrin (HPβCD), DMβCD appears to be the only cyclodextrin derivative known to date which, used as an absorption enhancing agent, gives rise to a complete absorption (virtually 100%) of nasally administered insulin.

For that matter, it has recently been demonstrated that dimethyl-β-cyclodextrin (DMβCD) can considerably improve the bioavailability of the nasally administered female sex

hormones estradiol and progesterone, and that DMβCD in the concentrations used has only a mild effect on the ciliary movement of human nasal epithelial tissue in vitro (Hermens et al., Pharm Res. 7 (1990) 500-503; Schipper et al., Int. J.

Pharm. 64 (1990) 61-66). Cyclodextrins are cyclical oligomers of 6, 7 or 8 glucose units, named α-, β- and γ-cyclodextrin, respectively. The inside of these cyclical structures has lipophilic properties, while the outside has a hydrophilic character. Therefore cyclodextrins are capable of increasing the water-solubility of lipophilic drugs such as estradiol and progesterone by means of the formation of inclusion complexes and thus increasing the nasal absorption of these substances.

For polypeptide drugs such as insulin, the absorption improving effect of cyclodextrins observed according to the invention must be based on other mechanisms. The formation of inclusion complexes with insulin is substantially precluded because insulin is a high-molecular and hydrophilic substance. Without wishing to be bound by such an explanation, we would suggest the increased transport of polypeptides via the nasal membrane barriers under the influence of cyclodextrins might reasonably be explained by the effects of cyclodextrins on the viscosity of the mucus layer, by an inhibition of the

formation of insulin aggregates, by an interaction with the epithelial membranes, and by inhibitory effects on proteases and peptidases in the nasal epithelial tissue (Verhoef et al.; Eur. J. Drug Metab. Pharmacokin. 15 (1990) 83-93).

Intranasal administration of insulin with DMβCD as an absorption enhancer, therefore, offers good prospects for the therapy of diabetes mellitus patients, also because DMβCD has only a mild effect on the ciliary movement of human nasal epithelial tissue (Hermens et al., Pharm. Res. 7 (1990)

500-503; Schipper et al., Int. J. Pharm. 64 (1990) 61-66).

Moreover, it can now be reasonably assumed that DMβCD may also have a favourable effect on the nasal absorption of other therapeutically interesting (poly) peptides and proteins.

Examples of such (poly) peptides and proteins, including fragments and analogues with agonistic or antagonistic

properties derived therefrom, are the following:

thyrotropin-releasing hormone, luteinizing hormone-releasing hormone, thyrotropin, luteinizing hormone, follicle

stimulating hormone, growth hormone, prolactin, chorionic gonadotropin, placental lactogen, adrenocorticotropic hormone, melanocyte-stimulating hormone, enkephalins, endorphins, dynorphins, dermorphins, kyotorphin, vasopressin, oxytocin, calcitonin, parathyroid hormone, cholecystokinin, glucagon, gastrin, secretin, pancreozymin, motilin, substance P,

bombesin, neurotensin, neurokinin, caerulein, kallikrein, bradykinin, angiotensin, renin, angiotensin converting enzyme inhibitors, atrial natriuretic peptides, brain natriuretic peptides, endothelins, urokinase, interferons, interleukins, tissue-derived plasminogen activator, growth factors (insulinlike growth factors, epidermal growth factor, transforming growth factor, platelet-derived growth factor, tumour necrosis factor), thymosin, thymopoietin, erythropoietin,

gammaglobulins, Factor VII, Factor VIII, peptide-type

antibiotics (bacitracin, colistin, gramicidin, polymyxin), peptide-type anti-tumour agents (bleomycin, neocarcinostatin). Accordingly, the invention also includes nasal preparations which comprise such drugs. Preferred embodiments, however, are nasal preparations containing insulin or calcitonin as the active ingredient.

The invention is not limited to nasal preparations and nasal administration, but more broadly comprises other forms of trans-mucosal administration, such as buccal, sublingual, rectal, vaginal and pulmonary administration. Nasal

preparations and nasal administration, however, are highly preferable.

The trans-mucosal preparations according to the invention will in practice contain a pharmacologically active amount of the active agents present therein. This amount will vary with the nature of the pharmacologically active substances and will further depend inter alia on the desired dose. Preparations in the form of an aqueous solution suitable for nasal

administration may for instance contain 0.01 to 10 mg/ml of active agent. As regards the amount of dimethyl-β-cyclodextrin, the preparations will contain an amount thereof that enhances the absorption of the active agent present. DMβCD concentrations of 0.01 to 10% (w/v) are options, although concentrations of 0.5 to 5% are preferable.

Naturally, the trans-mucosal preparations according to the invention may also contain one or more adjuvants

conventionally used in trans-mucosal preparations, such as preservatives, stabilizers, etc. Agents suitable for these and other purposes are known to anyone skilled in the art.

The invention will now be illustrated in and by the following examples.

Example 1

(1) Preparation of nasal insulin formulations

Insulin solutions were prepared by dissolving human insulin powder (20 IU/mg) in physiological salt solution containing 5mM HCl. The solutions obtained were then

neutralized with 0.1M NaOH to final insulin concentrations of 1 mg/ml.

For the nasal administration the following cyclodextrins in final concentrations of 5% (w/v; weight percent per volume unit) were added to the insulin solutions mentioned:

α-cyclodextrin (α-CD), γ-cyclodextrin (γ-CD), hydroxypropyl-β-cyclodextrin (HPβCD) and dimethyl-β-cyclodextrin (DMβCD).

β-cyclodextrin (β-CD) was added in a final concentration of 1.8% (w/v), since this percentage is the maximum amount of β-CD which can be dissolved in aqueous media. The cyclodextrins used are commercially available products. For intravenous administration the insulin solution was diluted 8 times with physiological salt solution to a final concentration of

0.125 mg/ml.

(2) Nasal absorption studies

In these studies the rat was used as an experimental animal model. Male Wistar rats (body weight approx. 200 g) were anesthetized with intramuscularly injected Hypnorm

(1 ml/kg). The femoral artery was provided with a cannula for taking blood samples during the nasal absorption studies of insulin. The trachea was also cannulated to prevent

respiration via the nose, and the oesophagus was tied to the trachea cannula to prevent swallowing of the nasal

formulation. Then by means of a microliter syringe 20μl of the insulin formulations mentioned, corresponding with an amount of 0.4 IU (20μg) insulin, was intranasally administered via one of the two nares. After intranasal application, via the cannulated femoral artery, blood samples of 0.3 ml were taken, 2, 5, 10, 15, 20, 30, 45, 60, and 120 minutes after

administration. Approx. 20 μl of these blood samples was used directly for assaying the glucose concentrations in the blood (by means of Haemo-Glucotest strips in combination with a Reflolux reflectance meter). Then the blood samples were processed into serum by means of centrifugation. Finally, the insulin concentrations in the serum were determined with a commercially available radioimmunoassay kit for insulin; the assay limit of the kit used was ≤2μU insulin/ml serum.

To enable the determination of the absolute

bioavailability of the nasal insulin formulations used, insulin was also administered via an intravenous injection. To that end, with a microliter syringe a volume of 20μl of the insulin solution, corresponding to an amount of 0.05 IU

(2.5 μg) insulin, was intravenously administered via the femoral vein, and the cannula was directly washed out with 0.2 ml physiological salt. Then blood samples were taken via the cannula of the femoral artery, and the blood glucose and serum insulin concentrations were assayed as described

hereinabove. (3) Assay of the insulin bioavailability and the hypoglycemic. responses

Both the serum insulin and the blood glucose

concentrations were graphically plotted as a function of time. The measured glucose levels were expressed as percentages of the initial blood glucose concentrations. Then the areas under the serum insulin concentration - time curves and the blood glucose concentration - time curves (AUC = area under the curve) were calculated by means of the linear trapezoidal rule. The insulin AUC values were corrected for the endogenous serum insulin concentrations by subtracting from these values the AUCs obtained after administration of placebo

formulations.

The absolute bioavailability (F) of intranasally

administered insulin was finally calculated according to the formula F= (AUC intranasal/AUC intravenous) x 100%.

Table I summarizes the results of the absorption studies of intranasally administered insulin in rats. Addition of β-cyclodextrin, γ-cyclodextrin or hydroxypropyl-β-cyclodextrin in final concentrations of 1.8%, 5% and 5%, respectively, to the nasal insulin formulation does not have any or only a marginal influence on the serum insulin and blood glucose concentrations. Addition of 5% α-cyclodextrin increases the nasal absorption of insulin, which is evident from an increase in the insulin AUC values and a decrease in the glucose AUC values. A maximum effect of 50% of the initial glucose

concentrations in the blood is achieved 1 to 2 h after

administration, and the absolute bioavailability of the nasal insulin formulation with 5% α-cyclodextrin is 27.7% (Table I).

Of all investigated cyclodextrins, however, dimethyl-β*cyclodextrin proves to be the only absorption enhancer which yields a virtually complete absorption of intranasally

administered insulin. Addition of 5% dimethyl-β-cyclodextrin to the insulin formulation results in remarkably increased AUC values for insulin and highly reduced AUC values for glucose. For this cyclodextrin derivative, 1 h after administration of the nasal formulation a maximum effect is achieved of approx. 25% of the initial blood glucose concentrations, which remains virtually constant during the course of the experiment. The absolute bioavailability of this insulin preparation with 5% dimethyl-β-cyclodextrin as an absorption enhancing agent is complete, i.e. 100% (see Table I). Table I. Insulin AUC values, glucose AUC values and insulin bioavailability (F) after intranasal

administration of the insulin in rats

Dose Route Type cyclodextrin AUC insulin AUC glucose F insulin (N)

(μg) and conc. (%, w/v) (μU. ml^-1.min.) (% of initial value %

x 10³)

2.5 intravenous - 5610 ± 1850 8.9 ± 1. .9 100 (5)

0 intranasal - 2922 ± 777 12.7 ± 1. .3 0 (5)

20 Intranasal α-cyclodextrin (5%) 8873 ± 2364* 8.8 ± 3, .1* 27.7 ± 11.5 (6)

20 Intranasal β-cyclodextrin (1.8%) 3553 ± 1144 12.8 ± 5 .5 2.9 ± 5.3 (4)

20 Intranasal γ-cyclodextrin (5%) 2074 14.9 0 (2)

20 Intranasal hydroxypropyl-β- 3172 14.2 1.2 (2) cyclodextrin (5%)

20 Intranasal dimethyl-β-cyclodextrin 26346 ± 7818**⁺ 4.8 ± 2.3**⁺ 108.9 ± 36.4 (6)

(5%)

All values are the mean ± standard deviation for the number of rats given in parentheses (N).

AUC is the area under the serum insulin or blood glucose concentration - time curve determined until

2 h after administration.

* Significantly different from nasal placebo administration : p<0.05 ) Wilcoxo

* λ " " " " " " : p<0.005 ) Rank

+ Significantly different from nasal administration of insulin with α-cyclodextrin: p<0.05 ) test

Example 2

(1) Preparation of nasal calcitonin formulations

Calcitonin solutions were prepared by dissolving salmon calcitonin powder (4000 IU/mg) in physiological saline solution to final calcitonin concentrations of 100 IU/ml.

For nasal administration, α-cyclodextrin (α-CD) or dimethyl-β-cyclodextrin (DMβCD) in a final concentration of 5%

(w/v) was added to the calcitonin solutions. (2) Nasal absorption studies

The rat was used as an experimental animal model. Male Wistar rats (body weight approx. 200 g) were prepared as described for the nasal insulin absorption studies (Ex. 1). Using a microliter syringe 20 μl of the calcitonin

formulations mentioned, corresponding to an amount of 2 IU salmon calcitonin, was intranasally administered via one of the two nares. After the nasal application, blood samples of 0.5 ml were taken via the cannulated femoral artery, 0, 30, 60, 120 and 180 min after administration. The blood samples were processed into serum. Then the serum calcium

concentrations were determined by atomic absorption

spectrophotometry.

(3) Assay of the hvpocalcaemic responses

Table II summarizes the results of the absorption studies of intranasally administered calcitonin in rats.

Table II. Serum calcium concentrations after intranasal

administration of salmon calcitonin (10 IU/kg) in rats

Time after Additive

administration

(min) no . additive α- CD ( 5% ) DMβCD (5%)

0 100 100 100

30 91± 9 91± 9 88±4

60 91±13 82±15 80±4

120 97± 7 73± 4 75±3

180 92± 6 70±10 69±5

240 98± 6 79± 8 74±6

All values are presented as percentages of the initial calcium concentrations (to = 100%) and are the mean ±SD of 4 or 5

rats. Administration of calcitonin results only in a slight decrease of serum calcium levels at 30 and 60 minutes.

However, addition of α-cyclodextrin or dimethyl-β-cyclodextrin in final concentrations of 5% to the nasal calcitonin

formulation leads to remarkably enhanced calcitonin

absorption, as evident from the observed strong hypocalcaemic responses: both cyclodextrins reduce the serum calcium

concentrations to 70% of their initials values at 3 h after administration.

Claims

1. A pharmaceutical preparation of a form and composition suitable for trans-mucosal, particularly nasal,

administration, which preparation comprises at least one

(poly)peptide or protein drug, or a fragment or analogue thereof, as an active agent, as well as dimethyl-β-cyclodextrin as an absorption enhancing agent.

2. A pharmaceutical preparation according to claim l, characterized in that the preparation comprises as an active agent at least one compound selected from the group consisting of insulin, thyrotropin-releasing hormone, luteinizing

hormone-releasing hormone, thyrotropin, luteinizing hormone, follicle stimulating hormone, growth hormone, prolactin, chorionic gonadotropin, placental lactogen, adrenocorticotropic hormone, melanocyte-stimulating hormone, enkephalins, endorphins, dynorphins, dermorphins, kyotorphin, vasopressin, oxytocin, calcitonin, parathyroid hormone, cholecystokinin, glucagon, gastrin, secretin, pancreozymin, motilin, substance P, bombesin, neurotensin, neurokinin, caerulein, kallikrein, bradykinin, angiotensin, renin, angiotensin converting enzyme inhibitors, atrial natriuretic peptides, brain natriuretic peptides, endothelins, urokinase, interferons, interleukins, tissue-derived plasminogen activator, growth factors (insulinlike growth factors, epidermal growth factor, transforming growth factor, platelet-derived growth factor, tumour necrosis factor), thymosin, thymopoietin, erythropoietin, gammaglobulins, Factor VII, Factor VIII, peptide-type antibiotics (bacitracin, colistin, gramicidin, polymyxin), peptide-type anti-tumour agents (bleomycin, neocarcinostatin).

3. A pharmaceutical preparation according to claim 1, characterized in that the preparation contains insulin as an active agent.

4. A pharmaceutical preparation according to claim 1, characterized in that the preparation contains calcitonin as an active agent.

5. A pharmaceutical preparation according to any one of claims 1-4, characterized in that the preparation has the form of an aqueous solution suitable for nasal administration, which contains a pharmacologically active amount of drug and an absorption enhancing amount of dimethyl-β-cyclodextrin.

6. A method of trans-mucosally, particularly nasally, administering a (poly) peptide or protein drug, or a fragment or analogue thereof, comprising administering a pharmaceutical preparation of a form and composition suitable for transmucosal, particularly nasal, administration, which contains dimethyl-β-cyclodextrin as an absorption enhancing agent.

7. A method according to claim 6, characterized in that the preparation comprises as an active agent at least one compound selected from the group consisting of insulin, thyrotropinreleasing hormone, luteinizing hormone-releasing hormone, thyrotropin, luteinizing hormone, follicle stimulating hormone, growth hormone, prolactin, chorionic gonadotropin, placental lactogen, adrenocorticotropic hormone, melanocytestimulating hormone, enkephalins, endorphins, dynorphins, dermorphins, kyotorphin, vasopressin, oxytocin, calcitonin, parathyroid hormone, cholecystokinin, glucagon, gastrin, secretin, pancreozymin, motilin, substance P, bombesin, neurotensin, neurokinin, caerulein, kallikrein, bradykinin, angiotensin, renin, angiotensin converting enzyme inhibitors, atrial natriuretic peptides, brain natriuretic peptides, endothelins, urokinase, interferons, interleukins, tissuederived plasminogen activator, growth factors (insulin-like growth factors, epidermal growth factor, transforming growth factor, platelet-derived growth factor, tumour necrosis factor), thymosin, thymopoietin, erythropoietin, gammaglobulins, Factor VII, Factor VIII, peptide-type antibiotics (bacitracin, colistin, gramicidin, polymyxin), peptide-type anti-tumour agents (bleomycin, neocarcinostatin).

8. A method according to claim 6, characterized in that the preparation contains insulin as an active agent.

9. A method according to claim 6, characterized in that the preparation contains calcitonin as an active agent.

10. A method according to any one of claims 6-9,

characterized in that the preparation has the form of an aqueous solution suitable for nasal administration, which contains a pharmacologically active amount of drug and an absorption enhancing amount of dimethyl-β-cyclodextrin.