Link: Pharm/Biotech Resources
Title: Therapeutic treatment and prevention of
infections with a bioactive material(s) encapuslated within a
biodegradable-bio-compatable polymeric matrix
United States Patent: 6,902,743
Issued: June 7, 2005
Inventors: Setterstrom; Jean A. (Alpharetta, GA); Tice; Thomas R. (Birmingham, AL); Jacob; Elliot (Silver Spring, MD); Reid; Robert H. (Kensington, MD); van Hamont; John (West Point, NY); Boedecker; Edgar C. (Crownsville, MD); Jeyanthi; Ramassubbu (Columbia, MD); Friden; Phil (Bedford, MA); Roberts; F. Donald (Dover, MA); McQueen; Charles E. (Olney, MD); Bhattacharjee; Apurba (Kensington, MD); Cross; Alan (Chevy Chase, MD); Sadoff; Jerald (Washington, DC); Zollinger; Wendell (Silver Spring, MD)
Assignee: The United States of America as represented by the Secretary of the Army (Washington, DC)
Appl. No.: 055505
Filed: April 6, 1998
Abstract
Novel burst-free, sustained release biocompatible and biodegrable microcapsules which can be programmed to release their active core for variable durations ranging from 1-100 days in an aqueous physiological environment. The microcapsules are comprised of a core of polypeptide or other biologically active agent encapsulated in a matrix of poly(lactide/glycolide) copolymer having a molar composition of lactide/glycolide from 90/10 to 40/60, which may contain a pharmaceutically-acceptable adjuvant, as a blend of uncapped free carboxyl end group and end-capped forms ranging to ratios from 100/0 to 1/99.
Description of the Invention
This invention relates to compositions comprising active core materials(s) such as biologically active agent(s), drug(s) or substance(s) encapsulated within an end-capped or a blend of uncapped and end-capped biodegradable-biocompatable poly(lactide/glycolide) polymeric matrix useful for the effective prevention or treatment of bacterial, viral, fungal, or parasitic infections, and combinations thereof. In the areas of general and orthopedic surgery, and the treatment of patients with infectious or chronic disease conditions, this invention will be especially useful to physicians, dentists and veternarians.
BACKGROUND OF THE INVENTION
Wounds characterized by the presence of infection, devitalized tissue,
and foreign-body contaminants have high infection rates and are difficult to
treat.
To prevent infection, in bone and soft tissues systemic antibiotics must be
administered within 4 hours after wounding when circulation is optimal. This
has been discussed by J. F. Burke in the article entitled "The Effective
Period of Preventive Antibiotic Action in Experimental Incisions and Dermal
Lesions", Surgery, Vol. 50, Page 161 (1961). If treatment of
bacterial infections is delayed, a milieu for bacterial growth develops
which results in complications associated with established infections. (G.
Rodeheaver et al., "Proteolytic Enzymes as Adjucts to Antibiotic Prophylaxis
of Surgical Wounds", American Journal of Surgery, Vol. 127, Page. 564
(1974)). Once infections are established it becomes difficult to
systemically administer certain antibiotics for extended periods of time at
levels that are safe and effective at the wound site. Unless administered
locally, drugs are distributed throughout the body, and the amount of drug
hitting its target is only a small part of the total dose. This ineffective
use of the drug is compounded in the trauma patient by hypoglycemic shock,
which results in a decreased vascular flow to tissues. (L. E. Gelin et al.,
"Trauma Workshop Report: Shockrheology and Oxygen Transport", Journal
Trauma. Bol. 10, Page 1078 (1970)).
Additionally, infections caused by multiple-antibiotic resistant bacterial
are on the up-swing and we are on the verge of a potential world-wide
medical disaster. According to the Centers for Disease Control, 13,300
patients died in U.S. hospitals in 1992 from infections caused by
antibiotic-resistant bacterial. Methicillin-resistant S. aureus (MRSA)
is rapidly emerging as the "pathogen of the 90's".
 | a. Some major teaching hospitals in the U.S. report that up to 40% of strains of S. aureus isolated from patients are resistant to methicillin. Many of these MRSA strains are susceptible only to a single antibiotic (vancomycin). |
| b. Should MRSA also develop resistance to vancomycin, the mortality rate among patients who develop MRSA infections could approach 80%, thereby increasing the threat of this infectious killer. |
Moreover, Vancomycin resistance is on the up-swing:
| a. 20% of Enterococci are now resistant to vancomycin |
| b. In 1989, only one hospital in New York City reported vancomycin-resistant Enterococci. By 1991, the number of hospitals reporting vancomycin resistance rose to 38. |
| c. transfer of vancomycin-resistant gene (via plasmid) has been shown experimentally between Enterococcus and S. aureus. |
Many major pharmaceutical companies around the world have either completely
eliminated or significantly reduced their research and development programs
in the area of antibiotic research. According to a 1994 report by the
Rockefeller University Workshop in Multiple Antibiotic Resistant Bacteria,
we are on the verge of a "medical disaster that would return physicians back
to the pre-penicillin days when even small infections could turn lethal due
to the lack of effective drugs."
Despite recent advances in antimicrobial therapy and improved surgical
techniques, osteomyelitis (hard tissue or bone infection) is still a source
of morbidity often necessitating lengthy hospitalization. The failure of
patients with chronic osteomyelitis to response uniformly to conventional
treatment has prompted the search for more effective treatment modalities.
Local antibiotic therapy with gentamicin-impregnated poly(methylmethacrylate)
(PMMA) bead chains (SEPTOPAL™, E. Merck, West Germany) has been utilized in
Germany for the treatment of osteomyelitis for the past decade and has been
reported to be efficacious inseveral clinical studies. The beads are
implanted into the bone at the time of surgical intervention where they
provide significantly higher concentrations of gentamicin than could
otherwise be achieved via systemic administration. Serum gentamicin levels,
on the other hand, remain extremely low thereby significantly reducing the
potential for nephro- and ototoxicity that occurs in some patients receiving
gentamicin systemically.
Since SEPTOPAL™ is not currently approved by the Food and Drug
Administration for use in the United States, some orthopedic surgeons in
this country are fabricating their own "physician-made beads" for the
treatment of chronic osteomyelitis. A major disadvantage of the beads,
however, is that because the PMMA is not biodegradable it represents a
foreign body and should be removed at about 2-weeks postimplantation thereby
necessitating in some cases an additional surgical procedure. A
biodegradable-biocompatable, antibiotic carrier, on the other hand, would
eliminate the need for this additional surgical procedure and may
potentially reduce both the duration as well as the cost of hospitalization.
The concept of local, sustained release of antibiotics into infected bone is
described in recent literature wherein antibiotic-impregnated PMMA
macrobeads are used to treat chronic osteomyelitis. The technique as
currently used involves mixing gentamicin with poly(methylmethacrylate) bone
cement and molding the mixture into beads that are 7 mm in diameter. These
beads are then locally implanted in the infected site at the time of
surgical debridement to serve as treatment. There are, however, significant
problems with this method. These include: 1) initially, large amounts of
antibiotics diffuse from the cement but with time the amount of antibiotic
leaving the cement gradually decreases to subtherapeutic levels; 2) the
bioactivity of the antibiotic gradually decreases; 3)
poly(methylmethacrylate) has been shown to decrease the ability of
polymorphonuclear leukocytes to phagocytize and kill bacteria; 4) the beads
do not biodegrade and usually must be surgically removed; and 5) the
exothermic reaction that occurs during curing of poly(methymethacrylate)
limits the method to the incorporation of only thermostable antibiotics (primarly
aminoglycosides). Nevertheless, preliminary clinical trials using these
beads indicate that they are equivalent in efficacy to longer term (4-6
weeks) administration of systemic antibiotics.
In many instances, infectious agents have their first contact with the host
at a mucosal surface; therefore, mucosal protective immune mechanisms are of
primary importance in preventing these agents from colonizing or penetrating
the mucosal surface. Numerous studies have demonstrated that a protective
mucosal immune response can best be initiated by introduction of the antigen
at the mucosal surface, and parenteral immunization is not an effective
method to induce mucosal immunity. Antigen taken up by the gut-associated
lymphoid tissue (GALT), primarily by the Peyer's patches in mice, stimulates
T helper cell (Th) to assist in IgA B cell responses or stimulates T
suppressor cells (Ts) to mediate the unresponsiveness of oral tolerance.
Particulate antigen appears to shift the response towards the (Th) whereas
soluble antigens favor a response by the (Ts). Although studies have
demonstrated that oral immunization does induce an intestinal mucosal immune
response, large doses of antigen are usually required to achieve sufficient
local concentrations in the Peyer's patches. Unprotected protein antigens
may be degraded or may complex with secretory IgA in the intestinal lumen.
In the process of vaccination, medical science uses the body's innate
ability to protect itself against invading agents by immunizing the body
with antigens that will not cause the disease but will stimulate the
formation of antibodies that will protect against the disease. For example,
dead organisms are injected to protect against bacterial diseases such as
typhoid fever and whooping cough, toxins are injected to protect against
viral diseases such as poliomyelitis and measles.
It is not always possible, however, to stimulate antibody formation merely
by injecting the foreign agent. The vaccine preparation must be immunogenic
that is, it must be able to induce an immune response. Certain agents such
as tetanus toxoid are innately immunogenic, and may be administered in
vaccines without modification. Other importantagents are not immunogenic,
however, and must be converted into immunogenic molecules before they can
induce an immune response.
The immune response is a complex series of reactions that can generally be
described as follows:
Most antigens only elicit antibiodies with assistance from the T cells and, hence, are known as T-dependent (TD). These antigens, such as proteins, can be processed by antigen presenting cells and thus activate T cells in the process described above. Examples of such T-dependent antigens are tetanus and diphtheria toxoids.
Some antigens, such as polysaccharides, cannot be properly processed by antigen presenting cells and are not recognized by T cells. These antigens do not require T cell assistance to elicit antibody formation but can activate B cells directly and, hence, are known as T-independent antigens (TI). Such T-independent antigens include H influenzae type by polyribosyl-ribitol-phosphate and pneumococcal capsular polysaccharides.
T-dependent antigens differ from T-independent antigens in a number of ways. Most notably, the antigens differ in their need to be administered in conjunction with an adjuvant (a compound that will nonspecifically enhance the immune response). The vast majority of soluble T-dependent antigens elicit only low level antibody responses unless they are administered with an adjuvant. It is for this reason that the standard DPT vaccine (diptheria, pertussis, tetanus) is administered with the adjuvant alum. Insolubilization of TD antigens into an aggregated form can also enhance their immunogenicity, even in the absence of an adjuvant. Golub E S and W O Weigle, J. Immunol. 102:389, 1969). In contrast, T-independent antigens can stimulate antibody responses when administered in the absence of an adjuvant, but the response is generally of lower magnitude and shorter duration.
Four other differences between T-independent and T-dependent antigens are:
| a) T-dependent antigens can prime an immune response so that a memory response can be elicited upon secondary challenge with the same antigen. Memory or secondary responses are stimulated very rapidly and attain significantly higher titers of antibody that are seen in primary responses. T-independent antigens are unable to prime the immune system for secondary responsiveness. |
| b) The affinity of the antibody for antigen increases with time after immunization with T-dependent but not T-independent antigens. |
| c) T-dependent antigens stimulate an immature or neonatal immune system more effectively than T-independent antigens. |
| d) T-dependent antigens usually stimulate IgM, IgG1, IgG2a, and IgE antibodies, while T-independent antigens stimulate IgM, IgG1, IgG2b, and IgG3 antibodies. |
These characteristics of T-dependent vs. T-independent antigens provide both
distinct advantages and disadvantages in their use as effective vaccines.
T-dependent antigens can stimulate primary and secondary responses which are
long-lived in both adult and in neonatal immune systems, but must frequently
be administered with adjuvants. Thus, vaccines have been prepared using only
an antigen, such as diptheria or tetanus toxoid, but such vaccines may
require the use of adjuvants, such as alum for stimulating optimal
responses. Adjuvants are often associated with toxicity and have been shown
to nonspecifically stimulate the immune system, thus inducing antibodies of
specificities that may be undesirable.
Another disadvantage associated with T-dependent antigens is that very small
proteins such as peptides, are rarely immunogenic, even when administered
with adjuvants. This is especially unfortunate because many synthetic
peptides are available today that have been carefully synthesized to
represent the primary antigenic determinants of various pathogens, and would
otherwise make very specific and highly effective vaccines.
In contrast, T-independent antigens, such as polysaccharides, are able to
stimulate immune responses in the absence of adjuvants. Unfortunately,
however, such T-independent antigens cannot stimulate high level or
prolonged antibody responses. An even greater disadvantage is their
inability to stimulate an immature or B cell defective immune system (Mond
J. J., Immunological Reviews 64:99, 1982) Mosier D E, et al., J. Immunol.
119:1874, 1977). Thus, the immune response to both T-independent and
T-dependent antigens is not satisfactory for many applications.
With respect to T-independent antigens, it is critical to provide protective
immunity against such antigens to children, especially against
polysaccharides such as H. influenzae and S. pneumoniae. With
respect to T-dependent antigens, it is critical to develop vaccines based on
synthetic peptides that represent the primary antigenic determinants of
various pathogens.
One approach to enhance the immune response to T-independent antigens
involves conjugating polysaccharides such H. influenzae PRP (Cruse J.
M., Lewis R. E. Jr. ed., Conjugate vaccines in Contributions to Microbiology
and Immunology, vol. 10, 1989) or oligosaccharide antigens (Anderson P W, et
al., J. Immunol. 142:2464, 1989) to a single T-dependent antigen such as
tetanus or diptheria toxoid. Recruitment of T cell help in this way has been
shown to provide enhanced immunity to many infants that have been immunized.
Unfortunately, only low level antibody titers are elicited, and only some
infants response to initial immunizations. Thus, several immunizations are
required and protective immunity is often delayed for months. Moreover,
multiple visits to receive immunization may also be difficult for families
that live distant from medical facilities (especially in underdeveloped
countries). Finally, babies less than 2 months of age may mount little or no
antibody response even after repeated immunization.
One possible approach to overcoming these problems is to homogeneously
disperse the antigen of interest within the polymeric matrix of
appropriately sized biodegradable-biocompatable microspheres that are
specifically taken up by GALT. Eldridge et al. have used a murine model to
show that orally-administered 1-10 micrometer microspheres consisting of
polymerized lactide and glycolide, (the same materials used in resorable
sutures), were readily taken up into Peyer's patches, and the 1-5 micrometer
size were rapidly phagocytized by macrophages. Microspheres that were 5-10
micrometers (microns) remained in the Peyer's patch for up to 35 days, where
as those less than 5 micrometers disseminated to the mesenteric lymph node (MLN)
and spleen within migrating MAC-1+ cells. Moreover, the levels of specific
serum and secretory antibody to staphyloccal enterotoxin B toxoid and
inactivated influenza A virus were enhanced and remained elevated longer in
animals which were immunized orally with microencapsulated antigen as
compared to animals which received equal doses of non-encapsulated antigen.
These data indicate that microencapsulation of an antigen given orally may
enhance the mucosal immune response against enteric pathogens. AF/R1 pili
mediate the species-specific binding of E. coli RDEC-1 with mucosal
glycoproteins in the small intestine of rabbits and are therefore an
important virulence factor. Although AF/R1 pili are not essential for E.
coli RDEC-1 to produce enteropathogenic disease, expression of AF/R1 to
produce enteropathogenic disease, expression of AF/R1 promotes a more severe
disease. Anti-AF/R1 antibodies have been shown to inhibit the attachment of
RDEC-1 to the intestinal mucosa and prevent RDEC-1 disease in rabbits. The
amino acid sequence of the AF/R1 pilin subunit has recently been determined,
but specific antigenic determinants within AF/R1 have not been identified.
In the current study we have used these theoretical criteria to predict
probable T or B cell epitopes from the amino acid sequence of AF/R1. Four
different 16 amino acid peptides that include the predicted epitopes have
been synthesized: AF/R1 40-55 as a B cell epitope, 79-94 as a T cell epitope,
108-123 as a T and B cell epitope, and AF/R1 40-47/79-86 as a hybrid of the
first eight amino acids from the predicted B cell epitope and the T cell
epitope. We have used these peptides as well as the native protein to
stimulate the in vitro proliferation of lymphocytes taken from the Peyer's
patch, MLN, and spleen of rabbits which have received introduodenal priming
with microencapsulated or non-encapsulated AF/R1. Our results demonstrate
the microencapsulation of AF/R1 potentiates the cellular immune response at
the level of the Peyer's patch, thus enhancing in vitro lymphocyte
proliferation to both the native protein and its linear peptide antigens.
CFA/I pili, rigid thread-like structures which are composed of repeating
pilin subunits of 147 amino acid found on serogroups 015, 025, 078, and 0128
of enterotoxigenic E. coli (ETEC) (1-4, 18). CFA/I promotes mannose
resistant attachment to human brush borders (5); therefore, a vaccine that
established immunity against this protein may prevent the attachment to host
tissues and subsequent disease. In addition, because the CFA/I subunit
shares N-terminal amino acid sequence homology with CS1, CFA/II(CS2) and
CFA/IV(CS4(4), a subunit vaccine which contained epitopes from this area of
the molecule may protect against infection with various ETEC.
Until recently, experiments to identify these epitopes were time consuming
and costly; however, technology is now available which allows one to
simultaneously identify all the T cell and B cell epitopes in the protein of
interest. Multiple Peptide synthesis (Pepscan) is a technique for the
simultaneous synthesis of hundreds of peptides on polyethylene rods (6). We
have used this method to synthesize all the 140 possible overlapping
octapeptides of the CFA/I protein. The peptides, still on the rods, can be
used directly in ELISA assays to map B call epitopes (6. 12-14). We have
also synthesized all 138 possible overlapping decapeptides of the CFA/I
protein. For analysis of T cell Epitopes, these peptides can be cleaved from
the rods and used in proliferation assays (15). Thus this technology allows
efficient mapping and localization of both B cell and T cell epitopes to a
resolution of a single amino acid (16). These studies were designed to
identify antigenic epitopes of ETEC which may be employed in the
construction of an effective subunit vaccine.
CFA/II pili consist of repeating pilin protein subunits found on several
serogroups of enterotoxigenic E coli (ETEC) which promote attachment
to human intestinal mucosa. We wished to identify areas within the CFA/I
molecule that contain immunodominant T cell epitopes that are capable of
stimulating the cell-mediated portion of the immune response in primates as
well as immunodominant B cell epitopes. To do this, we (a) resolved the
discrepancy in the literature on the complete amino acid sequence of CFA/I,
(b) immunized three Rhesus monkeys with multiple i.m. injections of purified
CFA/P subunit in Freund's adjuvant, (c) synthesized 138 overlapping
decapeptides which represented the entire CFA/I protein using the Pepscan
technique (Cambridge Research Biochemicals), (d) tested each of the peptides
for their ability to stimulate the spleen cells from the immunized monkeys
in a proliferative assay (e) synthesized 140 overlapping octapeptides which
represented the entire CFA/I protein, and (f) tested serum from each monkey
for its ability to recognize the octapeptides in a modified ELISA assay. A
total of 39 different CFA/I decapeptides supported a significant
proliferative response with the majority of the responses occurring within
distinct regions of the protein (peptides beginning with residues 8-40,
70-80, and 126-137). Nineteen of the responsive peptides contained a serine
residue at positions 2, 3, or 4 in the peptide, and a nine contained a
serine specifically at position 3. Most were predicted to be configured as
an alpha helix and have a high amphipathic index. Eight B cell epitopes were
identified as positions 3-11, 11-21, 22-29, 32-40, 38-45, 66-74, 93-101, and
124-136. The epitope at position 11-21 was strongly recognized by all three
individual monkeys, while the epitopes at 93-101, 124-136, 66-74, and 22-29
were recognized by two of the three monkeys.
Recent advances in the understanding of B cell and T cell epitopes have
improved the ability to select probably linear epitopes from the amino acid
sequence using theoretical criteria. B cell epitopes are often composed of a
string of hydrophilic amino acids with a high flexibility index and a high
probability of turns within the peptide structure. Prediction of T cell
epitopes are based on the Rothbard method which identifies common sequence
patterns that are common to known T cell epitopes or the method of Berzofsky
and others which uses a correlation between algorithms predicting
amphipathic helices and T cell epitopes.
SUMMARY OF THE INVENTION
This invention relates to active core materials such as biologically active
agent(s), drug(s), or substance(s) encapsulated within a biodegradable-biocompatable
polymeric matrix. In view of the enomorus scope of this invention it will be
presented herein as Phases I, II, and III. Phase I illustrates the
encapsulation of antibiotics within a biodegradble-biocompatable polymeric
matrix for the prevention and treatment of wound infections. Phase II
illustrates the encapsulation of antigens (including oral-intestinal vaccine
antigens) within a biodegradable-biocompatable polymeric matrix against
diseases such as those caused by enteropathogenic organism. Phase III
illustrates the use of a biodegradable-biocompatible polymeric matrix, as
the delivery system, for burst-free programmable sustained release of
biologically active agents, inclusive of peptides, over a period of up to
100 days in an aqueous physiological environment.
Controlled drug delivery from a biodegradable-biocompatable matrix offers
profound advantages over conventional drug/antigen dosing. Drugs/antigens
can be used more effectively and efficiently, less drug/antigen is required
for optimal therapeutic effect and, in the case of drugs, toxic side effects
can be significantly, reduced or essentially eliminated through drug
targeting. The stability of some drugs/antigens can be improved allowing for
a longer shelf-life, and drugs/antigens with a short half-life can be
protected within the matrix from destruction, thereby ensuring sustained
release of active agent over time. The benefit of a continuous sustained
release of drug/antigen is beneficial because drug levels can be maintained
within a constant therapeutic range and antigen can be presented either
continuously or in a pulsatile mode as required to stimulate the optimal
immune response. All of this can be accomplished with a single dose of
encapsulated drug/antigen.
This invention contemplates, but is not limited to, medically acceptable
methods for the effective local delivery of biologically active agents that,
of themselves, are directly (e.g. drugs, such as antibiotics) or indirectly
(e.g. vaccine antigens) therapeutic or prophylactic. It also includes
drugs/agents that elicit/modulate natural biological activity.
Wounds characterized by the presence of infection, devitalized tissue, and
foreign-body contaminants have high infection rates and are difficult to
treat. This invention describes antibiotic formulation encapsulated within
microspheres of a biodegradble-biocompatable polymer that, when applied
locally to contaminated or infected wounds, provides immediate, direct, and
sustained (over a period up to 100 days), high concentrations of antibiotic
in the wound site (soft tissue and bone). By encapsulating antibiotics and
applying them directly, one can achieve a significant reduction in
nonspecific binding of the drug to body proteins, a phenomena commonly
observed following conventional systemic administration of free drugs.
Thus, less drug is required, higher concentrations are maintained at the
site of need, and efficacy is enhanced. This approach provides superior
treatment over conventional systemic administration of antibiotics for wound
infections because higher bacteriocidal concentrations can be achieved and
maintained in the wound environment. Higher concentrations kill more
bacteria. Applicants' invention for this application is described in Phase
I. Furthermore, applicants reasoned that a protective mucosal immune
response might be best initiated by introduction of an antigen at the
mucosal surface, because unprotected protein antigens delivered in a free
form may be degraded or may complex with secretory IgA in the intestinal
lumen precluding entry and subsequent processing in local immune cells. The
formulation of microspheres containing antigen small enough in size to be
phagocytized locally in the gut was envisioned as being able to induce an
elevated localized immune response. Applicants' invention for this
application is described in Phase II. In summary, applicants propose using
several methods for the local application of drugs including: 1) the direct
application of the encapsulated drug to a surgical/traumatized area, 2) oral
delivery that provides either local deposition of microencapsulated
antigen/drugs at mucosal membranes or transport across these membranes to
provide local adherence of microencapsulated drugs/antigen to mucosal
membranes to provide sustained release of drug/antigen into such tissue or a
body cavity, and/or 3) sustained intercellular or extracellular drug/antigen
release following subcutaneous injection.
In those instances where antibiotics are administered locally, applicants
have found that the controlled release of the antibiotic from within a
biodegradable-biocompatable polymeric matrix within 14 days to about 4 weeks
without significant drug trailing is especially useful. However, if desired,
the release of a biologically active agent from a polymeric matrix comprised
of an active agent and a blend of uncapped and end-capped biodegradable poly
DL(lactide-co-glycolide), can be controlled over a period of 1 to about 100
days without significant drug dumping or trailing. Such novel biocompatable-biodegradable
microspheres developed with a burst-free programmable sustained release of
biologically active agents, inclusive of polypeptides, are described in
applicants' U.S. patent application Ser. No. 08/590,973 filed Jan. 24, 1996.
When antibiotics are administered systemically in the conventional manner,
or locally as contemplated by the applicants, the immune response to the
antibiotic and the potential for hypersensitivity and/or anaphylactoid
response (especially to beta-lactam antibiotics such as penicillins/ampicillin)
is a clinical concern. In early studies the inventors observed a specific
IgG response to ampicillin as it was released from the microencapsulated
formulation (illustrated in the histogram, FIGS. 1 and 2). The response is
reminiscent of antibody elicited by vaccine antigens in conventional
vaccines. The response to vaccine antigens is known to be accentuated by the
use of an adjuvant such as alum. Alum is a crude, less adaptable delivery
vehicle than its counterpart, the biodegradble-biocompatable poly
DL(lactide-co-glycolide), of this invention—the polymeric matrix. This
knowledge stimulated additional studies relevant to the effects of sustain
release of agents on the immune response.
There are, in general, two forms of localized delivery which can be achieved
with PLGA microspheres-delivery which is localized to individual cell's of
the body (intracellular delivery); and delivery which is localized to
tissues within a specific region of the body (localized extracellular
delivery).
Applicants have prepared antibiotic and hepatitis vaccine formulations which
functioned by delivering localized extracellular doses of their active
agents. This was achieved by using relatively large microspheres which
served as a depot for the drug or antigen. Their large size 40-100 microns
in diameter precluded their being phagocytized or diffusing throughout the
intercellular fluid compartments of the body. Their drug agent loads were
thus released within their immediate vicinity which resulted in the
generation of very high local concentrations of antibiotic or the release of
sufficiently high concentrations of free antigen to induce an immune
response.
The large-diameter antibiotic bearing microspheres were originally developed
by applicants primarily for topical application on exposed debrided tissues
of combat wounds. However, an inherent property exhibited by the antibiotics
when topically applied to a wound site is the generation of measurable
levels of immune response. This concept of local delivery by topical
application of microspheres to tissue to achieve localized concentrations of
therapeutic agents was subsequently applied to the development of an oral
vaccine for protection against traveler's dierrhea caused by E. coli.
Vaccine antigen was encapsulated into microspheres whose diameters were
predominantly in the 5-10 micron size range based on an understanding that
microspheres of this size would not readily be either phagocytized or
transported across the gut wall into the body. Ingestion of these
microspheres thus constituted a localized delivery achieved by topical
application of the spheres to the wall tissue of the gut. This topical
application resulted in the localized trapping of a small percentage of
these sphere into the Peyer's patches where the spheres proceeded to release
their antigen in a localized fashion to immune cells located within the
intestinal Patches.
The concept of localized sustained local delivery has been further extended
to the delivery of analgesics and anesthetics to exposed dental pulp to
control pain and inflammatory responses. Again, the PLGA microsphere used
for this type of delivery are relatively large (40-100 um in diameter) and
serve as a topical depot for localized extracellular release of the drug.
Consistent with their understanding of the inherent immunogenic properties
exhibited by active core materials in vivo, applicants have moved on to
other non-topical application methods of using their microsphere delivery
system. Some of these center on the use of small diameter microspheres
ranging from sub micron to under 5 microns in diameter. These spheres allow
intracellular targeting of drug or antigen. They also allow for transmucosal
delivery of drugs or antigens. The concept of localized delivery in these
instances refers to the localized delivery of drug or agent within
individual target cells of the body regardless of their location or
distribution within the body. This approach is useful in development of
antitubercular, antimalarial, antiviral, and antichlamydial formulations
against intracellular parasites. It is also useful for the development of
vaccines against intracellular parasites and for direct delivery of agents
to presenting cells of the immune system.
Another nontopical application method of using PLGA microspheres resides in
their usefulness as injectable depots for drugs intended for either
localized or systemic delivery. Typically larger diameter microspheres are
used for depots as these are less likely to diffuse away. The local or
systemic nature of these delivery systems is, in part a function of the
release rate of the drug from the depot and the diffusional and solubility
characteristics of the drug being released. Cancer chemotherapeutics,
systemic antibiotics, delivery of antibiotics to infected bonare are
potential application of this system. Additional this non-topical systemic
depot application can be extended to the intravenous iv injection of
cancer-agent laden microspheres to embolize and destroy a malignant tumor.
Additionally, the PLGA microspheres can be used as a carrier to deliver
substances useful for the in modification of cells or genes in
bioengineering or genetic procedures.
Interest in the concept that antigens encapsulated within a biodegradable-biocompatable
polymeric matrix could be formulated as a vaccine with superior efficacy
over conventional vaccines, originated from the inventors' own observations
that the drug, ampicillin, when sustain released from poly
DL(lactide-co-glycolide) elicited antibody production. In these studies, the
applicants were able to measure specific IgG antibodies to free ampicillin
and to ampicillin released from microencapsulated ampicillin formulations in
the sera of mice previously "treated" with the ampicillin formulations using
ELISA. Numerous other studies also document the ability of beta-lactam
antibiotic to elicit antibody. Selected, more recent studies whose findings
are consistent with earlier discoveries made by applicants when conducting
experiments with ampicillin include those by Klein et al. (1993) whose
detected specific IgG antibodies (IgG and IgG3 subclasses) to the B-lactam
ring in patients receiving penicillin therapy, work by Nagakura, et al.
(1990) which detected specific antibodies to cephalexin, a B-lactam
antibiotic in the sera of guinea pigs, and Auci et al. (1993) who detected
benzyl penicilloyl specific IgM, IgG IgE, and IgA antibody forming cells in
lumphoid cells of mice given benzyl penicilloyl-Keyhole Limpet Hemocyanin.
Pharmaceutical compositions of antigens encapsulated with poly
DL(lactide-co-glycolide) are described in Phase II. The microspheres of the
invention allow for introduction of vaccine antigens to mucosal surfaces in
particles that can be subsequently taken up locally by phagocytic cells.
Such an approach for both drugs and antigens provides significant advantages
in potency and efficacy over conventional systemically administered drugs or
vaccines. A partial list of biologically active agents or drugs that will
potentially derive significant medical benefits from this delivery system
includes: antibacterial agents; peptides; polypeptides; antibacterial
peptides; antimycobacterial agents; antimycotic agents; antiviral agents;
antiparastic agents; antifungal; antiyeast agents; hormonal peptides;
cardiovascular agents; hormonal peptides; cardiovascular agents; narcotic
antagonists; analgesics; anesthetics; insulins; steroids including HIV
therapeutic drugs (including protease inhibitors) and AZT; estrogens;
progestins; gastrointestinal therapeutic agents; non-steroidal
anti-inflammatory agents; parasympathoimetic agents; psychotherapeutic
agents; tranquilizers; decongestants; sedative-hypnotics; non-estrogenic and
non-progestional steroids; sympathomimetic agents; vaccines; vitamins;
nutrients; and-migrain drugs; electrolyte replacements; ergot alkaloids; and
anti-inflammary agents; prostaglandins; cytoxic drugs; antigens; antibodies;
enzymes; growth factors; immumodulators; pheromones; prodrugs; prohormones;
psychotropic drugs; nicotine; antiblood clotting drugs; appetite
suppressants/stimulants and combinations thereof; contraceptive agents
include estrogens such as diethyl silbestrol; 17-beta-estradiol; estrone;
ethinyl estradiol; mestranol; progestins such as norethindrone; norgestryl;
ethynodiol diacetate; lynestrenol; medroxyprogesterone acetate;
dimethisterone; megestrol acetate; chlormadinone acetate; norgestimate;
norethisterone; ethisterone; melentate; norgestimate; norethisterone;
ethisterone; melengestrol; and spermicidal compounds such as
nonyphenoxypolyoxyethylene glycol; benzethonium chloride; chlorindanol;
include gastrointestinal therapeutic agents-such as aluminum hydroxide;
calcium carbonate; magnesium carbonate; sodium carbonate and the like;
non-steroidal. antifertility agents; parasympathomimetic agents;
psychotherapeutic agents; major tranquilizers such as chloropromaquine HCl;
clozapine; mesoridazine; metiapine; reserpine; thioridazine; minor
tranquilizers such as chlordiazepoxide; diazpam; meprobamate; temazepan and
the like; rhinological decongestants; sedative-hypnotics such as codeine;
phenobarbital; sodium pentobarbital; sodium secobarbital; other steroids
such as testosterone and testosterone propionate; sulfonmides;
sympathomimetic agents; vaccines; vitamins and nutrient such as the
essential amino acids; essential fats; anti-HIV agents; including AZT;
antimalarials such as 4-aminoquinolines; 8 aminoquinolines; pyrimethamine;
anti-migraine agents such as mazindol; phentermine; anti-Parkinson agents
such as L-dopa; antispasmodics such as atropine; methscopolamine bromide;
antispasmodics and anticholingeric agents such as bile therapy; digestants;
enzymes and the like; antitussives such as dextromethorphan and noscapine;
bronchodialtors; cardiovascular agents such as anti-hypertensive compounds;
Rauwolfia alkaloids; coronary vasodilators; nitroglycerin; organic nitrites;
pentaerythriotetranitrate; electrolyte replacements such as potassium
chloride; ergotalkalodis such as ergotamine with and without caffein;
hydrogenated ergot alkaloids; dihydroergocristine methanesulfate;
dihydroergocornine methanesulfonate; dihydroergokroyptine methaneusulfate
and combinations thereof; alkaloids such as stropine sulfate; Belladonna;
hyoscine hydrobromide; analgesics; narcotics such as codeine;
dihydrocodienone; meperidine; morphine; non-narcotics such as salicylates;
aspirin; caffeine; nicotine; acetaminophen; and d-propoxyphene; antibiotics
such as the cephalosporins including ceflacor and cefuroxime;
chloranphenical; gentamicin; Kanamycin A. Kanamycin B; the penicillins;
ampicillin; amoxicillin; streptomycin A; antimycin A; chloropamtheniol;
metromidazole; oxytetracyline penicillin G; the tetracyclines; including
minocycline; fluoro-quinolones including ciprofloxacin; ofoxacin; macrolides
including clarithromycin; frythromycin; aminoglycosides including gentamicin;
amikacin; tobramycin and kanamycin; beta-lactams including ampicillin;
polymyxin-B; amphotercin-B; aztrofonam; chloramphenicol; fusidans;
lincosamides; metronidazaole; nitro-furantion; imipenem/cilastin; quinolones;
systemic antibodies including rifampin; polygenes; sulfonamides;
trimethoprim; glycopeptides including vancomycin; teicoplanin and imidazoles;
anti-cancer agents; including anti-kaposi's sarcoma; agents and taxol anti-convulsants
such as mephenytoin; phenobarbital; trimethadione; anti-emetics such as
triethylperazine; antihistamines such as chlorophinazine; dimenhydrinate;
diphenhydramine; perphenazine; tripelennamine and the like;
anti-inflammatory agents such as hormonal agents; hydrocortisone;
prednisolone; prednisone; non-hormonal agents; allopurinol; water-soluble
hormone drugs; antibiotics; antiumor agents; anti inflammatory agents;
antipyretics; analgesics; such as acetaminophen, acetylsalicylic acid, and
the like; anesthetics such as lidocaine, xylocaine, and the like; anorexics
such as dexedrine, phendimetrazinetartrate, and the like; antiarthritics
such as methylprednisolone, ibuprofen, and the like; antiasthmatics such as
terbutaline sulfate, theophylline, ephedrine, and the like; antibiotics such
as sulfisoxazole, penicillin G, ampicillin, cephalosporins, amikacin,
gentamicin, tetracyclines, chloramphenicol, erytromycin, clindamycin,
isoniazid, rifampin, and the like; antifungals such as amphotericin B,
nystatin, ketoconazole, and the like; antivirals such as acyclovir,
amantadine, and the like; anticancer agents such as cyclophosphamide,
methotrexate, etretinate, and the like; anticoagulants such as heparin,
warfarin, and the like; anticonvulsants such as phenytoin sodium, diazepam,
and the like; antidepressants such as isocarboxazid, amoxapione, and the
like; antihistamines such as diphenhydramine HCl, chlorpheniramine maleate,
and the like; hormones such as insulin, progestins, estrogens, corticoids,
glucocorticoids, androgens, and the like; tranquilizers such thorazine,
diazepam, chlorpromazine HCl, reserpine, chlordiazepoxide HCl, and the like;
antispasmodics such as belladonna alkaloids, dicyclomine hydrochloride, and
the like; vitamins and minerals such as essential amino acids, calcium,
iron, potassium, zinc, vitamins A, B12, C, D and E, and the like;
cardiovascular agents such as prazosin HCl, nitroglycerin, propranolol HCl,
nydralazine HCl, verapamil HCl, and the like; enzymes such as lactase,
pancrelipase, succinic acid dehydrogenase, and the like; peptides and
proteins such as LHRH, somatostatin, calcitonin, growth hormone, growth
releasing factor, angiotensin, FSH, EGF, vasopressin, ACTH, human serum
albumin, gamma globulin, and the like; prostaglandins; nucleic acids;
carbohydrates; fats; narcotics such as morphine, codein, and the like;
psychoterapeitucs; anti-malarials; L-dopa, diuretics such as furosemide,
spironolactone, and the like; antiulcer drugs such as ranitidine HCl,
cimetidine HCl and the like, antitussives; expectorants; sedatives; muscle
relaxants; antiepileptic agents; antidepressants; antiallergic drugs;
cardiotonics; antiarrhythmic drugs; vasodilators; antihypertensives;
diuretics; anticoagulants; and antinaroctics; in the molecular weight range
of 100-100,000 daltons; indomethacin; phenylbutazone; prostaglandins;
cytotoxic drugs such as thiotepa; chloramucil; cyclosphosphamide; melphala;
nitrogen mustard; methotrexate; antigens such as proteins; glycoproteins;
synthetic peptides; carbohydrates; synthetic polysaccharides; lipids;
glycolipids; lipopolysaccharides (LPS); synthetic lipopolysaccharides and
with or without attached adjuvants such as synthetic muramyl dipeptide and
with or without attached adjuvants such as synthetic muramyl dipeptide
derivatives; antigens of such microorganisms as Neisseria gonorrhea;
Mycobacterium tuberculosis, Picarinii Pnfumonia; Herpes virus (humonis
types 1 and 2); Herpes zoster; Candidia albicans; Candida tropicals;
Trichomonas vaginalis; Haemophilus vaginalis; Group B streptoccoccus
ecoli; Microplasma hominis; Hemophilus ducreyi; Granuloma inguimale;
Lymphopathia venerum; Treponema palidum; Brucela aborus Brucela meitensis
Brucela suis; Brucella canis Campylobacter fetus; Campylobacer fetus
intesinalis; Leptospira pomona. Listeria monocytogens; Brucella ovis;
Equine herpes virus 1; Equine arteritis virus; IBR-IBP virus; Chlamydia
psittaci; Trichomonas foetus; Taxoplasma gondii; Escherichia coli;
Actinobacillus equuli; Salmonella abortus ovis. Salmonella abortus eui;
Pseudomonas aeruginosa; Corynebacterium equi; Corynebacterium pyogenes;
Actinobaccilus seminis; Mycoplasma bovigenitalium, Aspergil us fumigatus;
Absidia ramosa; Trypanosoma equiperdum; Babesia cabali; Clostridium tetani;
antibodies which counteract the above microorganisms; and enzymes such as
ribonuclease; neuramidinase; trypsin; glycogen phosphorylase; sperm lactic
dehydrogenase; sperm hyaluronidase; adenossinetriphosphase; alkaline
phosphatase; alkaline phospha esterase; amino peptides; typsin chymotrypsin
amylase; muramidase; acrosomal proteinase; dieterase; glutamic acid
dehydrogense; succunic and dehydrogenase; beta-glycophosphatase lipase; ATP-ase
alpha-peptate gamma-glutamyiotranspeptidase; sterold-beta-ol-dehydrogenase;
DPN-di-aprorase; and combinations thereof.
Immunological agents that can be encapsulated by this method include,
interleukins, interferon, colony stimulating factor, tumor necrosis factor,
and the like; allergens such as cat dander, burch pollen, house dust mite,
grass pollen, and the like; antigens of such bacterial organisms as
Streptoccus poneumoniae, Haemophilus influenzae, Staphylococcus aureus,
Streptococcus pyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes,
bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium
perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus
mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,
Bordetella pertussis, Francisella tularensis, Yersinia pestis, vibrio
cholerae, legionella pheumophila, Mycobacterium tubercolosis, Mycobacterium
leprae, Treponema pallidum, Leptspriosis interrogans, borrelia burgdorferi,
Campylobacer jejuni; and the like; antigens of such viruses as smallpox,
influenza A and B, respiratory syncytial, parainfluenza, measles, HIV,
varicella-zoster, herpes simplex 1 and 2, cytomeglavirus, Epstein-Barr,
rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps,
rabies, rubella, coxsackieviruses, equine encephalitis, Japanese
encephalitis, yellow fever, Rift Valley fever, lymphocytic choriomeningitis,
hepatitis B, and the like; antigens of such fungal, protozoan, and parasitic
organisms such as Cryptococcuc neoformans, Histoplasma capsulatum,
Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia
ricketsil, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci,
chlamydial trachomatis, plasmodium falciparum, Trypanosoma brucei, Entamoeba
histolytica, Toxaplasma gondii, Trichomonas vaginalis, Schistosoma mansoni,
and the like. These antigens may be in the form of whole killed organisms,
peptides, proteins, glycoproteins, carbohydrates, or combinations thereof.
Having generally described the invention; a further understanding can be
obtained by reference to certain specific examples which are provided herein
for purpose of illustration only and are not intended to be limiting unless
otherwise specified. Moreover; the polymeric matrix of this invention may be
used for the in situ production and controlled release of products that are
produced by the controlled release of encapsulated reactants. Additionally;
effective testing or monitoring devices for chemical agents or bioactive
agents can be made by encapsulating reagents which react as they are
released from the polymeric matrix, with agents sought to be detected. The
novel delivery system of this invention is applicable to all categories of
active substances capable of being used for the prevention and/or treatment
of human, animal and plant diseases. This delivery system is also applicable
to the design of novel diagnostic tests. Additionally, it can be useful for
the delivery to a subject of a polyfunctional mixture or cocktail
formulation of encapsulated active (i.e. biologically) substances for the
prevention and/or treatment of diseases of the same or different origin. The
encapsulated formulation ingredients would be comprised of multiple drugs,
multipe vaccines or a combination thereof.
Applicants contemplate that the invention will be useful in the formulation
of disease specific compositions for the prevention and/or treatment of
diseases and/or ailments which include: viral infections; bacterial
infections; fungal infections; yest infections; parastic infections and more
specific diseases and/or ailments; such as, aids; alzheimer's dementia;
angiogenesis diseases; aphthour ulcers in AIDS patients; asthma; atopic
dermatitis; psoriasis; basal cell carcinoma; benign prostatic hypertrophy;
blood substitute; blood substitute in surgery patients; blood substitute in
trauma patients; breast cancer; cutaneous & metastatic; cachexia in AIDS;
campylobacter infection; cancer; pnemonia; sexually transmitted diseases
(STDs); cancer; viral diseases; candida albicians in AIDS and cancer;
candidiasis in HIV infection; pain in cancer; pancreatic cancer; parkinson's
disease; peritumoral brain edema; postoperative adhesions (prevent);
proliferative diseases; prostate cancer; ragweed allergy; renal disease;
restenosis; rheumatoid arthritis; rheumatoid arthritis; allergies; rotavirus
infection; scalp psoriasis; septic shock; small-cell lung cancer; solid
tumors; stroke; thrombosis; type I diabetes; type I diabetes w/kidney
transplants; type II diabetes; viseral leishmaniasis; malaria; periodontal
or gum disease; cardiac rthythm disorders; central nervous system diseases;
central nervous system disorders; cervical dystonia (spasmodic torticollis);
choridal neovascularization; chronic hepatitis A, B and C; colitis
associated with antibiotics; colorectal cancer; coronary artery thrombosis;
cryptosporidiosis in AIDS; cryptosporidium parvum diarrhea in AIDS; cystic
fibrosis; cytomegalovirus disease; depression; social phobias; panic
disorder; diabetic complications; diabetic eye disease; diarrhea associated
with antibiotics; erectile dysfunction; genital herpes; graft-vs host
disease in transplant patients; growth hormone deficiency; head and neck
cancer; head trauma; stroke; heparin neutralization after cardiac bypass;
hepatocellular carcinoma; HIV; HIV infection; huntington's disease; CNS
diseases; hypercholesterolemia; hypertension; inflammation; inflammation and
angiogensis; inflammation in cardiopulmonary bypass; influenza; migrain head
ache; interstitial cystitis; kaposi's sarcoma; kaposi's sarcoma in AIDS;
lung cancer; melanoma; molluscum contagiosum in AIDS; multiple sclerosis;
neoplastic meningitis from solid tumors; non-small cell lung cancer; organ
transplant rejection; osteoarthritis; rheumatoid arthritis; osteoporosis;
drug addiction; shock; ovarian cancer; and pain.
Also contemplated here are those diseases or health conditions capable being
benefitted by the list of biologically active agents or drugs previously
listed in the Summary of the Invention.
Effects of Microencapsulated Antibiotics on the Immune Responce
Preclinical studies evaluating microencapsualted antibiotics in animals have
demonstrated that targeted local release of antibiotics directly into
infected soft tissue and bone via sustained release of the drug from poly
DL(lactide-co-glycolide) will greatly enhance antibiotic efficacy for both
prophylaxis and treatment. Antibiotic hypersensitivity was, from the
beginning, the most obvious untoward clinical concern of this novel approach
to antibiotic delivery. What effect would sustained antibiotic release have
on the hypersensitive patient?Prior to the filing of applicants' parent
application Ser. No. 590,308 on Mar. 16, 1984, which disclosed the local
application of encapsulated antibiotics to treat wound infection, it was
commonly known that an inherent property of free antibiotics such as
ampicillin, is that they elicit an immune response in man and induce the
production of antibodies. Thus, interest in the immune response elicited
from the sustained release of immunogens intensified in order to capture the
beneficial aspects of this immunogenic event in a manner which would advance
the frontiers of medical science. This led to additional studies with
sustain released antibiotics and led the inventors to postulate that
antigens encapsulated in lactide/glycolide could potentially provide a more
effective method of active immunization than free antigen alone. The recent
report in "Vaccination onto Bare Skin", by De-chu Tang, et al., Nature, vol.
388, August 1977, of the success with gene-based vaccines by topical
application to the skin continues to support position that an inherent
property of topically applied antibiotics is that they elicit an immune
response in man and induce the production of antibiodies. In follow on
experiments, vaccine antigens were encapsulated and studies were performed
to explore this hypothesis as illustrated in Phase II, herein.
Claim 1 of 154 Claims
1. A composition for the burst-free, sustained, programmable release of
active material(s) over a period from 1-100 days, which comprises: (1) an
active material and (2) a carrier which may contains
pharmaceutically-acceptable adjuvant, comprised of a blend of uncap and
end-capped biodegradable-biocompatible copolymer wherein said composition
comprises a capacity to completely release histatin in an aqueous
physiological environment within from 1 to 40 days with a 99/1 blend of
uncapped and end-capped poly(lactide/glycolide) having a L/G ratio of
48/52 to 52/48, and a molecular weight less than 15,000.
____________________________________________
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