Vitamin B12 —biodegradable micro particulate conjugate...

Details Vitamin B12 —biodegradable micro particulate conjugate... Vitamin B12 —biodegradable micro particulate conjugate...

2001-03-01

2002-11-19

Park, Hankyel T. (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Conjugate or complex

C514S052000

Reexamination Certificate

active

06482413

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a complex for oral delivery of drugs, therapeutic protein/peptides, vaccines, which are loaded in Vitamin B
12
coupled particulate carriers system with spacers in between. Typically, VB
12
acts as ligand for endocytosis through intestine and the particulate systems will act as a cargo protecting the intestinal labile drugs to deliver them to systemic destination and/or to the specific site required.
BACKGROUND ART
Although many peptide/proteins, pharmaceuticals and vaccines are currently administered by injection, this method of delivery has a number of disadvantages which has led the scientific community to strive to develop an alternative oral delivery system for these bioactives. The inherent limitations to parenteral delivery include (1) patient compliance (there is a need for repeated injections due to the short half-life of these molecules) (2) discomfort from this form of delivery caused by the need for repeated prolonged dosage regimen (3) highly variable bioavailability both within and between subjects for molecules such as subcutaneous insulin and (4) the non-physiological delivery pattern particularly of subcutaneous injections. Further, small increases in actual drug delivered due to changes in dosage and/or mode of delivery may cause down-regulation of the desired response to most of these bioactives. In contrast most often a pulsatile or flat delivery profile is required which mimics the normal physiological rhythm. Apart from the problem described, parenteral vaccines are of limited efficacy due to the need for repeated vaccination and due to the fact that they elicit only humoral immunity.
To address the above problems, various non-invasive delivery systems have been attempted. The major delivery barriers common to these routes are (1) poor intrinsic permeability due to large size and hydrophilicity of the bioactive (2) enzymatic degradation in the hostile environment of GIT by lumenal proteases and cellular peptidases, unlike some of the traditional drugs. Although these odds are formidable for the per oral route, it enjoys advantages in terms of convenience and patient compliance as well as safety, less stringent quality control, cost of therapy and for vaccines, the potential for unlimited frequency of boosting. Furthermore, oral vaccines offer the potential to protect against not only enteric pathogens (by producing localized sIgA), but also a wide range of pathogens infecting other mucosa (respiratory and genital) by producing a common disseminated mucosal immune response. Furthermore oral vaccines may prove particularly useful in the elderly because mucosal immunity, unlike systemic immunity does not seem to be an age associated dysfunction. This mode of immunization may also be beneficial in the very young, because mucosal immunity develops earlier in ontogeny than systemic immunity. Over the past few decades significant efforts have been made to develop oral protein/vaccine systems using permeation enhancers (ZOT, which has proven to be toxic on long term use), enzyme inhibitors, surfactants, emulsions, colon targeted systems, bioadhesive systems and particulate systems. Although the challenges to develop oral systems are numerous, the potential therapeutic need remains high, particularly with increased identification of novel peptides and increased production of existing protein drugs from the biotechnology arena.
Peptide/protein drugs and vaccines are mostly given by parenteral routes. The problems for the oral delivery are:
1) Degradation of the above bioactives in the harsh environment of intestine and by gut proteases.
2) Their large size and hydrophilicity causes permeability problem across the intestine.
3) The fragility of these bioactives precludes to be formulated as oral dosage forms.
4) And finally their short in-vivo half lives.
Over the past 3-4 decades efforts have been made to deliver them by oral route using various formulation approaches such as emulsions, microspheres, nanoparticles, vasicular carriers such liposomes, using permeation enhancers and protease inhibitors and by protein-carrier conjugates.
Recently it has been shown that the problem of poor intrinsic permeability can be possibly overcome by delivery via a specific carrier mechanism that transports pharmaceuticals from the intestine into the circulation. In this regard, Russell-Jones and co workers [1994] have found the possibility of coupling these bioactives with Vitamin B12 (VB
12
) in a manner that does not interfere with intrinsic factor (IF) mediated uptake of the VB
12
carrier to the systemic circulation. Some interesting results have been achieved with oral VB
12
conjugates of LHRH (Russell-Jones 1998), interferon (Habberfield and Jensen-Pippo PPO, 1996) G-CSF and erythropoietin (Habberfield, 1996). However, such successful oral delivery of conjugates cannot be obtained with many other bioactives because of the limited uptake of VB
12
(1 nmol/dose), the loss of bioactivity due to covalent linkage, loss of IF affinity of VB
12
(steric factor) and finally the liability of such conjugates to GIT degradation. To address the above problems we have made endeavors to make an oral delivery system (VB
12
-sphere conjugate) that could be targeted to the systemic circulation through VB
12
-IF-IFR ligand-mediated endocytosis via ileocytes of the intestine following oral administration. In vitro transport of VB
12
coated polystyrene nanoparticles (non-biodegradable, hydrophobic) has been demonstrated across Caco-2 cell cultures (Russell Jones, 1997, 1999). However, the level of transport cannot be extended to other polymeric particulates in general. This can be explained by the differing physicochemical characteristics of polymers such as hydrophobicity, as hydrophobic materials may be more readily taken up by cell surface lipid bilayers. Also one of the prerequisites for drug delivery is that the polymer used to make the nanoparticles should readily or slowly be degraded in the systemic environment or by enzymes which release the pharmaceutical molecule and thereby initiate its bioactive response. Hence, the use of nonbiodegradable polymers for transport studies is not necessarily indicative, of uptake by other biodegradable polymers in general. Further, developing a system of loading of hydrophilic peptide/protein or vaccine bioactives into hydrophobic polymeric carriers is definitely not obvious.
Among all these approaches, there was some enhancement of delivery, but major break through of achieving therapeutically relevant dose delivery is not achieved. In the recent past, a delivery system was invented by one of the co-inventors using VB
12
as carrier molecule which is coupled with protein drug to be delivered i.e. VB
12
-protein conjugates. VB
12
binds with intrinsic factor of the intestine (VB
12
+IF→VB
12
IF) in the duodenum and the whole VB
12
IF complex binds to intrinsic factor receptor (IFR) at the ileum of the small intestine. From there, it is endocytosed and subsequently transcytosed by transcobolamin (TcII) to reach systemic circulation.
Therefore, if protein is coupled with VB
12
, it will be co-transported along with VB
12
molecule to reach systemic circulation.
Two conditions are to be satisfied for this approach i.e.
Case 1: VB
12
molecule must retain its IF affinity after being coupled to protein to be delivered.
Case 2: Also protein molecule must retain its bioactivity after coupling to VB
12
.
For case 1: Native VB
12
(cynocobolainin) cannot be directly coupled to protein, i.e. VB
12
losses its IF affinity by such coupling.
Therefore, in the above technology VB
12
is hydrolyzed, where it forms 3 isomers (a, b and e isomers). Among the 3 isomers, the ‘e’ isomer retains considerable IF affinity after coupling to other molecules.
Case 2: For the ease of coupling to provide suitable groups and to retain full bioactivity of protein drug, various derivatives of e-VB
12
using various spacers. These derivatives are coupled to protein drugs to be delivered. Such spacers also inc

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