OA21005A - Combretum micranthum extracts for the effectiveness of β-lactam antibiotics. - Google Patents

Abstract

The invention relates to extracts of Combretum micranthum used in combination with antibiotics of the β-lactam family (cephalosporins). These extracts are used as a natural inhibitor in the fight against bacterial resistance by producing enzymes (broad-spectrum β-lactamases). Obtaining the inhibitor involves the steps of harvesting, obtaining the extracts and formulating the substance. It can have three galenic forms, namely oral, injectable and cutaneous.

Description

The present invention relates to extracts based on Combretum micranthum. It is advantageously used in the fight against bacterial resistance through the production of enzymes (β-lactamases) in association with antibiotics from the β-lactam family.

As it stands, antibiotics since their discovery have saved the lives and improved the health of countless people and undoubtedly constitute one of the greatest successes of modern medicine. They have unfortunately been victims of their success: their repeated and sometimes abusive administration has in fact led to the emergence of strains of bacteria which are resistant to them. With the emergence of resistance, antibiotics are less and less effective, and as a result the treatment of infections today becomes complicated. Indeed, certain strains of bacteria appear insensitive to several antibiotics, or even to almost all of the available antibiotics, which can lead to a therapeutic impasse. Because of resistance, treatments are longer, several antibiotics are prescribed, and patients suffer more side effects. The appearance of resistance concerns almost all antibiotics developed. This is a real “arms race” between humans and bacteria, to the extent that resistance systematically appears after the introduction of a new antibiotic.

Today, bacterial resistance to antibiotics has become one of the most important problems in anti-infectious therapeutics worldwide and in the pharmaceutical industry. Infections caused by antibiotic-resistant bacteria are responsible for a high rate of morbidity and mortality compared to infections caused by antibiotic-susceptible bacteria.

On April 30, 2014, the World Health Organization (WHO) published its first global report on antimicrobial resistance, including antibiotic resistance, which highlights that "this serious threat is no longer a prediction, but a reality in every region of the world, and that everyone, whatever their age and country, can be affected.” Adding that, if nothing is done, the world is moving towards a post-antibiotic era, "where common infections and minor injuries that have been treated for decades could kill again."

The first comprehensive analysis of the global impact of antimicrobial resistance (AMR) estimates that resistance itself caused 1.27 million deaths in 2019; more deaths than major killers like HIV/AIDS or malaria and antimicrobial-resistant infections played a role in 4.95 million deaths (Howard, 2022).

β-lactam antibiotics represent the main family of antibiotics most developed and used in the world, thanks to their broad spectrum of action, their low toxicity, their effectiveness and their low cost for certain molecules (Livermore, 1995) .

Bacteria have developed various mechanisms to counteract the action of these β-lactams, the most common of which is the synthesis of enzymes (β-lactamases) which inactivate β-lactams by chemical modification (Walsh, 2003). β-lactamases effectively and irreversibly catalyze the hydrolysis of the amide bond of the β-lactam cycle of antibiotics of the β-lactam family, giving a product that is biologically inactive, i.e. one which completely loses its antimicrobial activity. (Matagne et al., 1998).

The role of bacterial enzymes in the development of resistance is rather versatile and involves several key mechanisms (Wright, 2005). Enzymes involved in cell wall biosynthesis, as well as the synthesis of nucleic acids and metabolites, serve as direct targets for antibiotics. The resistance mechanism is associated with structural modifications of these enzymes. Another mechanism is associated with the enzymatic modification of structural elements affected by antibiotics: for example, the modification of ribosomes by methyltransferases. A large group of enzymes modifies or destroys the structure of antibiotics by inactivating them, it is this group that we are interested in. Bacterial enzymes that determine resistance generally belong to large superfamilies; many of them came from enzymes that originally had other functions (Morar and Wright, 2010). The genes responsible for the synthesis of these enzymes and their mutational variability are often located on mobile genetic elements, thus ensuring the rapid spread of resistance between microorganisms. Indeed, the destruction or modification of the antibiotic structure is one of the most common mechanisms of resistance involving enzymes. Depending on the type of reactions they catalyze, the enzymes involved in this resistance mechanism are subdivided into hydrolases, transferases and oxidoreductases. We focused mainly on Hydrolases, more precisely Beta-lactamases. β-Lactamases hydrolyze the amide bond in the β-lactam ring, the common structural element of all β-lactam antibiotics (penicillins, cephalosporins, carbapenems, and monobactams). They form a superfamily of enzymes that currently has more than 2,000 members (Bonomo, 2017). According to amino acid sequence homology, β-lactamases are subdivided into four molecular classes (Hall and Barlow, 2005). Enzymes of classes A, C and D are serine hydrolases, enzymes of class B are metalloenzymes. Serine β-lactamases have similar structural elements to the C domain of PBPs, indicating that they are evolutionarily related (Ghuysen, 1994). The evolution of β-lactamase develops via two main mechanisms: the emergence of new mutations in the genes of known enzymes and the emergence of enzymes with a new structure. The high mutation rate of β-lactamases and the localization of their genes on mobile genetic elements contribute to the rapid spread of resistant bacteria, which pose a global threat (Bush and Jacoby, 2010). Bacteria carrying up to eight β-lactamase genes simultaneously have been detected (Bush, 2013).

Class A β-lactamases (CTX-M, TEM, SHV, and KPC lactamases) are the most common (Bush, 2013). Mutational variability is a characteristic of TEM and SHV βlactamases. Key active site mutations increase the volume of the enzyme and make it capable of hydrolyzing the bulk molecules of second to fourth generation cephalosporins (Orencia et al., 2001). These mutant forms are known as extended-spectrum β-lactamases (ESBLs). Some mutations of amino acid residues located distant from the active site are compensatory and can have multidirectional effects on stability (Grigorenko et al., 2018). Class C β-lactamases efficiently hydrolyze cephalosporins. Initially, this class was represented by enzymes encoded by chromosomal genes and having an inducible type of expression. Then, enzymes encoded by genes located on mobile elements were discovered (Philippon et al., 2002). Class D β-lactamases include OXA-type β-lactamases and are the most structurally diverse enzymes among the serine β-lactamases. Molecular class B is a heterogeneous family of metallo-βlactamases (MBL) (Bebrone, 2007). They contain one or two zinc ions in their active site, hydrolyze almost all β-lactam antibiotics except monobactams, and are inhibited by chelating agents (EDTA, dipicolinic acid and o -phenanthroline). The emergence of new MBL variants (e.g., NDM-type carbapenemases) and their co-expression with serine β-lactamases results in the emergence of bacteria resistant to all β-lactam antibiotics (Nordmann et al., 2011).

In order to overcome the resistance induced by the production of β-lactamases, an active search for inhibitors of these enzymes is currently underway (Docquier and Mangani, 2018) (King et al., 2016). In clinical practice, combinations of β-lactams with clavulanic acid, sulbactam and tazobactam (which contain a βlactam ring, form a more stable acyl-enzyme complex and have a low deacylation rate) are used intensively. to inhibit class A enzymes. Newer inhibitors that are structurally similar to β-lactams but do not contain a β-lactam ring include the diazabicyclooctanes (avibactam and MK-7655). They form carbamyl-enzyme complexes with involvement of the catalytic serine, which are then subjected to slow and reversible recyclization, accompanied by the release of an inhibitory molecule. These inhibitors have proven effective against class A, C and partly D β-lactamases. Boronic acid derivatives capable of inhibiting class A carbapenemases are widely studied.

Some studies have demonstrated resistance of enterobacteria to certain existing inhibitors (clavulanic acid) (Amana et al., 2019), for this the discovery of inhibitors having good penetration at the membrane level of Gram-negative bacteria would also be a a big step in the fight against resistance to enterobacteria, more precisely, extended-spectrum beta-lactamase (ESBL)-producing enterobacteria.

Medicinal plants constitute an important reserve of inhibitory molecules which could be very effective against beta-lactamase-producing bacteria.

In this context, the inventors have found a new inhibitor of resistance enzymes. In particular, the inventors have demonstrated that extracts of Combretum micranthum are capable of restoring the activity of a class of common antibiotics of the β-lactam family against bacterial strains presenting resistance by production of enzymes. (Beta-lactamases). This inhibitor can therefore be used in combination with antibiotics from the β-lactam family whose effectiveness is weakened, due to the resistance presented by enterobacteria through enzyme production.

Experimental part

Resistance profile of enterobacteria strains

Clinical strains of enterobacteria collected at the Saint Camille Hospital of Ouagadougou (HOSCO) and the Tengandogo University Hospital Center (CHUT) were used for resistance tests. The following antibiotics were tested: amoxicillin/clavulanic acid (AUG), Cefotaxime (CTX), ceftriaxone (CRO), Ceftazidime (CAZ), aztreonam (ATM), and imipenem (IMF).

Production of beta-lactamases by enterobacteria strains

The production of Beta-lactamases of our resistant Enterobacteriaceae strains has been proven by the classic Polymerase Chain Reaction (PCR) method, using primers specific to Beta-lactamase genes. Several types of beta-lactamase genes have been identified, confirming the resistance of these strains through enzyme production.

Inhibition of beta-lactamases by Combretum micranthum

Combretum micranthum extraction protocol

The different organs (Leaves and Roots) of our plant A were pulverized using an aluminum mortar. 15 to 75 grams of powder were extracted with methanol (150 to 350 ml) by maceration for 48 hours at 37° C. with mechanical stirring at 200 rpm. The filtrates were concentrated using a rotary evaporator equipped with a vacuum pump, collected in petri dishes and dried in an oven at 37° C. These extracts obtained were used for phytochemical tests and biological.

Preparation of Combretum micranthum extracts

Fifty (50) mg of the extract were weighed and added to a volume of DMSO 99.9% of between 3 and 10 ml. The mixture was homogenized to dissolve the extract and obtain a concentration between 5 and 20 mg/ml. From the previous solution we made a 1/10 dilution. The solution obtained has a concentration of between 0.5 and 5 mg/ml with 1% DMSO. From the previous solution, another dilution was made and we obtained a final solution with a concentration between 50 and 200 pg/ml.

Preparing the antibiotic solution

An antibiotic stock solution was prepared by weighing 40 to 70 mg of powdered antibiotic. It was dissolved in 4 to 7 ml of sterile distilled water to obtain an initial concentration between 5000 and 15,000 pg/ml, from which the different antibiotic concentrations were prepared. All manipulations were carried out under sterile conditions either under the hood or next to the flame.

Inhibitory activity of Combretum micranthum extracts on a resistant strain of enterobacteria by enzyme production

Previously we determined the highest concentration of the antibiotic used for the in vitro tests which did not inhibit the growth of bacteria. We then selected a strain resistant to all antibiotics and carrying at least two resistance genes for testing with Combretum micranthum extracts.

This is how, from a pre-culture of a strain called U29, inoculations on liquid medium were carried out. To determine the presence of an inhibitor in Combretum micranthum, the following conditions were used:

❖ Tube 1: Negative control = LB alone <♦ Tube 2: Positive control = LB + U29 strain ♦: · Tube 3: LB + U29 strain + Antibiotic

Tube 4: LB+ Combretum micranthum extract 10 to 30 ug of extracts of

Combretum micranthum ❖ Tube 5: LB+ Combretum micranthum extract + Antibiotic ❖ Tube 6: LB+ Combretum micranthum extract _{30 to 60 ug of extracts of} , _A Combretum micranthum * Tube 7: LB+ Combretum micranthum extract + Antibiotic

The initial optical densities (OD) of all tubes were read at 630 nm in triplicate from the spectrophotometer, and the tubes were incubated at 37°C for 18 to 24 h. The final optical densities were also read again at 630 nm in triplicate.

Enzymatic Kinetics

Extraction of β-lactamases

In gram-negative bacteria, β-lactamases are normally secreted and maintained in the periplasm. In practice, the crude extracts carried out for analytical purposes (demonstration and measurement of β-lactamase activity) concerned a volume of between 2 and 10 mL of bacterial suspension. The bacterial pellet obtained after centrifugation of between 1000 and 5000 revolutions per minute for 30 minutes at 4°C in the centrifuge was suspended with 300 μl of phosphate buffer comprised of 50 mM and 100 mM pH 7. The contents of the periplasmic space were released by the physical method of freezing/thawing cycles (Bidwell and Reeves, 1980). The principle of the method consists of preparing a cold bath by cooling acetone with dry ice. A liquid medium is obtained which is at a temperature ranging from -65°C to -75°C. The cells are immersed for 8 minutes in this medium, which results in instant freezing. Then they are transferred to a bain-marie at 37°C for instant defrosting. These sudden changes in state (frozen state - thawed state and vice versa) cause pulverization of the bacterial walls with the release of beta-lactamases which are localized in the periplasmic compartment of the bacteria. The samples are then centrifuged at 8000 rpm for 20 minutes. Cell debris is then removed and the supernatants collected and tested for β-lactamase activity. The supernatant is stored at -20°C.

Hydrolysis of Nitrocefin and β-lactams by crude extracts of β-lactamases

The different bacterial extracts obtained were tested with a view to measuring their β-lactamase activity with Nitrocefin.

This chromogenic β-lactam is an all-purpose substrate for almost all β-lactamases. A reaction medium consisting of 50mM phosphate buffer pH7, enzyme (5 to 10μΙ) and Nitrocefin in final concentration of ΊΟΟμΜ is produced in a quartz cuvette.

The extracts exhibiting β-lactamase activity were tested with different βlactams in order to determine their hydrolytic profile. For this purpose, an antibiotic solution at a concentration of 2 mM was prepared in Eppendorf tubes. This solution is used as a substrate in a reaction medium where the final concentration of antibiotic was 25μΜ. Enzymatic activities were monitored on a BioTek EL808 spectrophotometer. The hydrolysis curves of the β-lactam nuclei by the different extracts were recorded at the appropriate wavelengths. All these experiments were carried out at 30°C in 50mM phosphate buffer pH 7.

The initial rate (Vo) of hydrolysis of each compound (expressed in absorbance units per second) was evaluated according to the relationship Vo = ΔΑΑ / At in which AAA / At represents the slope at the origin of the variation curve absorbance at wavelength λ as a function of time. The Vo obtained and expressed in absorbance units/s is convertible into μΜ of transformed substrates or products released by the relation Vo = AAA / At x 1 / Δελ in which Δελ is the difference between the molar extinction of the product and the substrate at wavelength A (Zeba et al., 2004).

Inhibitory activity of Combretum micranthum extracts on β-lactamases

The activity of β-lactamases was determined in the presence and absence of extracts from plants A and B. For this, in order to determine the hydrolytic profile of β lactamases, a final reaction volume of 500 μΙ was prepared, composed of Buffer phosphate (50 μΜ pH 7), Enzyme (5-20ul) and antibiotic. The ODs were read immediately at the appropriate wavelengths every 10 seconds for 5 min.

For the determination of the inhibition of β-lactamases by the Combretum micranthum extract, a final reaction volume of 500 μΙ was prepared, composed of phosphate buffer (50 μΜ pH 7), Enzyme (5-20ul) and 5-20 μΙ of plant extract (Leaf and Bark). The mixture was incubated at 37°C for 30 min. The antibiotic which is the substrate was then added, then the ODs were read at the appropriate wavelengths every 10 seconds for 5 min.

The inhibition rate was determined according to the formula:

Vo without inhibitor ^- Y0 with inhibitor

Inhibition rate =-------------------------------------- χ Ιθθ

V0 without inhibitor

Vo without inhibitor: Vo (Phosphate buffer + enzyme + CRO)

Vo with inhibitor: Vo (Phosphate buffer + enzyme -(-Combretum micranthum extract + CRO)

Results

Resistance profile of enterobacteria

These strains showed resistance to all antibiotics tested (Figure 1).

Figure 1: Resistance profile of one of the strains studied

Resistance Genes

Resistance genes by enzyme production were detected in the enterobacteria studied (PER, GES, SFO, CTX-M, SHV, VIM and IMP).

Activity of Combretum micranthum on a strain of enterobacterium producing beta-lactamases

The enterobacteria strain resistant to concentrations of 300pg/ml of antibiotic was used for the tests. Tests in the test tubes in the presence of the negative/f control and positive tubes showed that Combretum micranthum extract inhibits the effect of beta-lactamases on antibiotics of the beta-lactam family (Figure 2).

Figure 2: Test for inhibition of an enterobacterium using Combretum micranthum extract

Legend: (a) = Negative control, LB alone; (b) =LB+U29, (c) = LB+U29+CTR, (d) = LB + Combretum micranthum extracts (25pg/ml, (e) = LB + CRO + Combretum micranthum extracts (25 pg/ml )

The optical densities before and after 18 to 24 hours of incubation at 37°C were read with a spectrophotometer. The results after incubation were recorded in Figure 3.

0.6 .............................................. 0.567

LB alone LB+U29 LB+U29+CTR LB+PBF LB+PBF+CTR

Condition of tubes

Figure 3: Optical densities obtained during the different study conditions for a concentration of Combretum micranthum extracts at 25 pg/ml

Legend: LB Only= Negative control, LB+U29= strain alone, LB+U29+CTR= strain+ 5 enterobacteria, LB+PBF= Extracts of Combretum micranthum + enterobacteria,

LB+PBF+CTR= Combretum micranthum extracts + enterobacteria+ antibiotic

The hydrolytic profile of beta-lactam antibiotics by beta-lactamases

The enzymes of the enterobacteria strains were extracted and analyzed with a spectrophotometer. The substrates used are: Nitrocefin which is a beta-lactamase test substrate and antibiotics.

The hydrolysis curves of Nitrocefin for some enzymatic extracts are shown in Figures 4, 5 and 6:

Absorbance 482 nm

Figure 4: Hydrolysis curve of Nitrocefin (CPR) by the enzymatic extract of

Klebsiella pneumoniae

Nitrocefin is a beta-lactamase test substrate. The enzymatic activity was measured according to the appearance of the product in the reaction medium, hence the growth of the curve.

Absorbance at 482 nm

80 100

Time in seconds

120 140 160

Figure 5: Hydrolysis curve of Nitrocefin (CPR) by the enzymatic extract of Escherichia coli

The enzymatic activity was also measured as a function of the appearance of the product in the reaction medium, hence the fact that the curve is also increasing.

Absorbance at 482 nm

Figure 6: Hydrolysis curve of Nitrocefin (CPR) by the enzymatic extract of 5 Stenotrophomonas maltophilia

The enzymatic activity was measured according to the appearance of the product in the reaction medium.

Once the activity of Beta-lactamases was confirmed with the test substrate (Nitrocefin), the hydrolytic profile of the antibiotics was studied. The hydrolysis curve of a 10 antibiotic is shown in Figure 7:

Abs CHU-ΤΙ

Figure 7: The hydrolysis curve of Ceftriaxone (CRO) by the enzymatic extract of Klebsiella pneumoniae in the absence of medicinal plant extract

The enzymatic activity was measured as a function of the disappearance of substrate 5 (antibiotic) in the reaction medium, hence the decrease in the curve.

The initial speed (Vo) of enzymatic hydrolysis without medicinal plant extract A is equal to 0.0006 AU/s (absorbance unit per second).

Inhibitory activity of Combretum micranthum extracts on β-lactamases

The extracts of Combretum micranthum showed good inhibitory activity on 10 β-lactamases as indicated in Figure 8.

Abs PBF

0.5

0.45 _ω 0.4 .g 0.3 · j s. — — ' “ ' “V—Q—

................

.A; y = 4E-05x + 0.2928........................

^! § 0.15: ....................................... ...... .................................. ^: -.... ...................................

0.05' - - - - .........................................

.................................................. .....—..................-.................... . —......-0 50 100 150 200

Time(s)

Figure 8: The hydrolysis curve of Ceftriaxone by the enzymatic extract of Klebsiella pneumoniae in the presence of the extract of Combretum micranthum leaves.

The enzymatic activity was measured as a function of the disappearance of the substrate (antibiotic) in the reaction medium, hence the decrease in the curve.

The initial velocity Vo is 0.00004 AU/s (unit of absorbance per second).

The inhibition rate of the leaf extracts was calculated by the following formula:

0.0006-0.00004

Inhibition rate = x 100

0.0006

The inhibition rate for extracts from the leaves is 93.33%.

Acute toxicity in vivo

To carry out the toxicity tests, we used twelve (12) NMRI strain mice, aged 8 to 12 weeks, including 6 males and 6 females with an average weight of 44.94 g and 30.37 g respectively. These animals were divided into two groups of 6 with regard to each sex: the test group and the control group. All the animals were weighed before being distributed into the different groups then fasted for 24 hours at the end of which they received for the test groups an extract solution concentrated at 2000 mg/kg using a gastric tube and the control groups received only distilled water. The animals were again deprived of food for four hours during which their behavior was observed, and during the 14 days of the experiment their water and food consumption, their weight variation and their behavior were monitored and recorded.

At the end of the 14 days, the animals were weighed just before their sacrifice which was done after anesthesia with an excess of Ketamine + Lidocaine.

No animal deaths were recorded during the experiment, the LDso of this plant is therefore greater than 2000 mg/kg.

The present invention thus aims to propose an inhibitor of resistance enzymes based on extracts of Combretum micranthum capable of restoring the activity of a class of usual antibiotics of the β-lactam family with respect to bacterial strains presenting resistance by production of enzymes (β-lactamases).

The process for obtaining the resistance enzyme inhibitor follows the following steps:

- 1st step: Harvesting the organs of Combretum micranthum

- 2nd step: Obtaining Combretum micranthum extracts

- 3rd step: Formulation of the substance of Combretum micranthum

Harvesting Combretum micranthum organs

The different organs of Combretum micranthum are harvested in all seasons, mainly before sunrise. It mainly concerns the leaves. These organs are then dried at room temperature in the shade.

Obtaining Combretum micranthum extracts

The dried leaves are then pulverized. Fifteen to seventy-five grams of powder were extracted with methanol (150 to 350 ml) by maceration for 48 hours at 37° C. with mechanical stirring at 200 rpm. The filtrates are concentrated using a rotary evaporator equipped with a vacuum pump, collected in petri dishes and dried in an oven at 37° C. 10 to 70 mg of the extract were weighed and added to a volume of DMSO 99.9% of between 3 and 10 ml. The mixture was homogenized to dissolve the extract and obtain a concentration between 5 and 20 mg/ml.

Formulation of Combretum micranthum substance

In its formulation, the following dosage forms for use in humans are considered:

1. Oral Form

The best compromise for good antibiotic activity of combinations of an antibiotic from the cephalosporin family and Combretum micranthum extracts is 500 pg / 25 pg, i.e., 500 pg of antibiotic and 25 pg for Combretum micranthum extracts.

Thus, the possible combinations with an antibiotic for the different doses are: Dried powder for suspension: administered to children • 125 mg Antibiotic + 6.25 mg of Combretum micranthum extract • 250 mg Antibiotic + 12.50 mg of Combretum micranthum extract Capsules: administered in adults • 250 mg Antibiotic + 12.50 mg Combretum micranthum extract • 500 mg Antibiotic + 25 mg Combretum micranthum extract

For dry powder for suspension, the following excipients can be used citric acid, sugar, sodium benzoate, xanthan gum, sunshine yellow, vanilla and aerosil, talc, sodium starch glycolate, aerosil, magnesium stearate, corn starch dry.

2. Injectable Form

The best compromise for good antibiotic activity of combinations of antibiotics and Combretum micranthum extracts is 500 pg / 25 pg, i.e., 500 pg of antibiotic and 25 pg for Combretum micranthum extracts.

Thus, the possible combinations with an antibiotic for the different doses are: • 250 mg antibiotic + 12.5 mg of Combretum micranthum extract administered in adults and children • 500 mg antibiotic + 25 mg of the extract of Combretum micranthum administered in adults and children • 1000 mg antibiotic + 50 mg of Combretum micranthum extract administered in adults

The present invention is particularly intended for the fight against bacterial resistance by production of enzymes in both humans and animals. It is in no way limited to the formulations and dosages described above. A person skilled in the art will be able to make any variation consistent with his spirit.

Claims (6)

1. Plant extract associated with antibiotics characterized in that it is obtained from different organs of Combretum micranthum to increase the effectiveness of antibiotics. 2. Plant extract associated with antibiotics according to claim 1, characterized in that it inhibits bacterial resistance enzymes at a rate of at least 93%. 3. Plant extract combined with antibiotics according to claims 1 and 2, characterized in that it is used in combination with an antibiotic from the β-lactam family. 4. Plant extract associated with antibiotics according to claim 3, characterized in that the antibiotic of the β-lactam family is third generation cephalosporin. 5. Plant extract associated with antibiotics according to the preceding claims, characterized in that it is active at antibiotic concentrations of 500 ± 200 pg/ml and of Combretum micranthum extract at concentrations of 25 ± 20 pg/ml. ml. 6. Plant extract associated with antibiotics according to the preceding claims, characterized in that the combination of the extract of Combretum micranthum and the antibiotic present three galenic forms for use in humans and in animals. namely the oral form, the injectable form and the cutaneous form.