Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2002-07-16
2004-07-20
Barts, Samuel (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S001110, C536S055100, C536S123100
Reexamination Certificate
active
06765091
ABSTRACT:
TECHNICAL SECTOR
The present invention relates to the field of the Medicine, in particular with the chemical synthesis of oligosaccharide mixtures derived of ribose-ribitol-phosphate, which are used as active principle in vaccines for the prevention of infections caused by
Haemophilus influenzae
type b (Hib), as well as with the vaccines containing said oligosaccharide mixtures.
PRIOR ART
Haemophilus influenzae
type b is a serious human health problem worldwide. The bacterium causes, mainly in children under the age of 5, meningitis, pneumonia, epiglotitis, and other diseases of the respiratory tract. The sequels observed, ranged from auditive problems until sever is mental retardation, attain in many countries more than 30% of the survivors from the disease. Recent estimates of World Health Organization indicated that more than 550 000 children dies annually from the diseases caused by
Haemophilus influenzae
type b in the world.
Purified capsular polysaccharide of
Haemophilus influenzae
is able to induced protective immunity in adults, however the immune response in children is very poor and practically absent in infants under 2 years old.
The capsular polysaccharide has the following structure:
It has been demonstrated that the main problem is the antigen's nature itself, due to being a polysaccharide, it is a T-independent antigen unable to stimulate infant immune system, still immature. There was demonstrated that the solution to this problem can be achieve by linking covalently (conjugating) the polysaccharide to a protein known as carrier. The product thus obtained, known as conjugated vaccine, induces antibody protecting level from two months of age.
Chu et al., (Infection immunity 1983, 40, 245-256) obtained the conjugate from the native capsular polysaccharide and tetanus toxoid after activation with cyanogen bromide.
Gordon (U.S. Pat. No. 4,496,538) activated the natural polysaccharide with cyanogen bromide and then conjugated it to difteria toxoid through the adippic acid dihidrazide.
Hilman et al. (U.S. Pat. No. 4,459,286) conjugated the natural polysaccharide through 6-aminocaproic acid to menongococcal outer membrane protein after their initial activation.
In all the previous conjugation processes, the covalent linkage takes place between several groups of the capsular polysaccharide and the carrier protein.
The ability to induce a protecting immunity in infants depends on the structure of the conjugate. When the conjugation is performed on the native polysaccharide, the groups participating in the linkage are randomly distributed alone the polysaccharide chain, making characterization of each batch very complex.
All these vaccines are very difficult to analyze by physic-chemical methods; therefore the common practice is the evaluation of each batch through studies of immunogenicity in experimental animals. However, the behavior of the conjugate is different in children than in experimental animals.
According to World Health Organization (WHO) criteria of stable quality (M. R. Holliday, C. Jones, Biologicals, 1999, 27, 51-53) the control of conjugated vaccines should be based on physic-chemical methods demonstrating similarity from one batch to another.
In order to facilitate this task, conjugated vaccines should be more and more defined at the molecular level. One alternative solution to this problem is the synthesis of capsular polysaccharide fragments. The process for the construction of the
Haemophilus influenzae
type b antigen by synthesis has two main steps, the synthesis of the disaccharide intermediate and its oligomerization. Several approaches have been developed for this synthesis.
Beuvery at al. (European patent application EPO 0276 516; U.S. Pat. No. 5,034,519; Tetrahedron Lett. 28, 1553, 1987) and Hoogerhout et al. (J. Carbohydr. Chem, 7, 1988, 399-416) obtained by synthesis a fragment of the capsular polysaccharide that was claimed as containing between 3 to 20 repetitive units. To achieve this goal the disaccharide intermediate 2 was first prepared and then through solid-phase chemistry or solution synthesis, an oligomer containing 6 repeating units was prepared (Elie et al., Rec. Trav. Chim. Pays-Bas 108, 1989, 219). The oligomers were conjugate to proteins or peptides through a spacer. The conjugate trimer was as immunogenic in mice than commercially available vaccine prepared from the capsular polysaccharide.
In a preferred embodiment illustrating synthetic pathway followed, the subsequent strategy was used: 1-synthesis of the ribitol, 2-coupling to ribose, 3-selective introduction of substituents in the ribose unit and 4-introduction of the phosphor-activating group. Using this pathway the key disaccharide derivative 2 was obtained in only 15 reaction steps.
The overall yield is 7% (Hermans et al., Recl. Trav. Chim. Pays-Bas 1987, 106, 498-504). Furthermore, this process has two major drawbacks: the process includes 11 chromatographic steps and the protecting groups of the key intermediary are not ideal for the oligomerization process.
In the oligomerization, or second step of the synthesis, the method used is solution synthesis through activation by phosphotriester, allowing yields between 70-90% per cycle, based on the disaccharide. The main drawback of such procedure is the impossibility of preparing fragments containing more than 4 repeating units, because the yields decrease dramatically. The disaccharide intermediate 2 has three different protective groups, making very difficult the deprotection of the final product. Therefore this disaccharide isn't the more convenient for oligomerization through solid-phase chemistry. Inasmuch the synthesis of an hexamer was reported.
In immunological assays (Peters CCAM, et al, Infect. Immunity 1991, 59, 3504-10) only it is reported the conjugation of a trimer to tetanus-toxoid and its immunogenicity in mice and monkeys.
G. Just, J. Upeslacis (European patent application EP 0 320 942, L. Chan; G. Just, Tetrahedron, 46, 1990, 151-162) synthesized also a fragment of the capsular polysaccharide through a disaccharide intermediate and synthesis in solution chemistry. With the aim of preparing the optimal intermediate for the synthesis of the antigen, a different pathway was selected: 1-synthesis of ribitol, 2-synthesis of the ribose unit with adequate protecting groups, 3-coupling of ribose and ribitol and 4-introduction of the phosphor active functionality.
Using this methodology a procedure for the intermediate 3 as phosphoramidite displaying a better selection of protecting groups for the oligomerization procedure. In order to attain this objective, a bigger number of steps were necessary. The key derivative was attained in 19 steps and with the use of 8 chromatography processes of purification.
The disaccharide 3 was oligomerized in solution giving fragments of the capsular polysaccharide containing 3 repeating units with yields based on disaccharide between 70-90% per cycle.
Kandil et al. (Syn. Lett., 1992, 555-7), Chon et al. (PCT patent application WO93/15205 y U.S. Pat. No. 5,679,352), synthesized a fragment of the capsular polysaccharide using the same disaccharide intermediate 3 and through solid-phase chemistry, using as support monomethoxypolyethylenglycol obtained fragments containing up to 6 repeating units, the yield per cycle was 95%.
Krivan, et al.. (PCT patent application WO94/00149) and Nilsson, et al. (J. Carbohydr. Chem. 10, 1991, 1-22) obtained a fragment of 10 repeating units with a similar disaccharide intermediate and solid-phase chemistry. This fragment was conjugated through a spacer to Hib adhesin. The phosphonate intermediate was obtained in 21 steps with an overall yield of 5%. At least 7 chromatographic steps were also needed. The oligomerization process was performed using H-phosphonates and solid-phase chemistry using Merrifield aminated resin. The antigen was obtained with incorporation of 97-99% per cycle.
Chiu Machado et al. (J. Carbohydr. Chem., 13 1994, 465-474) and Cuban patent 22424 reported an efficient procedure for the synthesis of a suitab
Bencomo Vicente Guillermo Vérez
Roy Rene
Barts Samuel
Krishnan Ganapathy
Lackenbach & Siegel LLP
Universidad de la Habana
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