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Tropical Journal of Pharmaceutical Research, Vol. 8, No. 6, December, 2009, pp. 501-508

Moustapha Ouédraogo^1*, Rasmané Semdé¹, Issa T Somé¹, Moussa Ouédraogo¹, Rasmata Ouédraogo¹, Viviane Henschel², Brigitte Evrard³, Jacques Dubois², Karim Amighi²and Innocent P Guissou¹

¹UFR - Sciences de la Santé, Université de Ouagadougou, 03 BP 7021 Ouagadougou 03, Burkina Faso, ²Pharmacy Institute, Université Libre de Bruxelles, Campus Plaine, Boulevard du Triomphe, B 1050 Bruxelles, ³Pharmacy Institute, Université de Liège, CHU Tour 4, Av. de l’Hôpital, B 4000 Liège 1, Belgium.

Purpose: Drugs that are administered by parenteral route must be apyrogenic. The aim of this study was to develop an in vitro endotoxin test for liquid crystalline gels for use as implants, using a monoolein–water liquid crystalline gel as a model.
Methods: The gel-clot technique was used. The gel was dissolved first in isopropyl myristate, and the endotoxins were extracted with water for bacterial endotoxin test. Tests for the labelled lysate sensitivity and interfering factors were performed to validate the developed method. The limit of detection of endotoxin in the gel was also determined.
Results: The labelled lysate sensitivity was confirmed. It was not influenced by the presence of extracts from the gels. Endotoxins in the contaminated test gels were completely extracted. Endotoxin concentration in the tested gels was below the calculated threshold endotoxin level.
Conclusion: A method to perform in vitro endotoxins test of liquid crystalline gels was successfully developed and validated. Application of the technique to gels currently being developed in our laboratories indicate that the gels were apyrogenic.

Keywords: In vitro bacterial endotoxin test; liquid crystalline gels; test validation; monoolein–water.

Drugs that are administered by the parenteral route must be apyrogen [1-3]. Consequently, it is necessary to detect and/or quantify endotoxins in parenteral products such as implants. Bacterial endotoxins can provoke in humans, fever, shock, or even death [4]. To verify if parenteral drugs are apyrogenic, tests performed in rabbits are broadly accepted. Nevertheless, both ethical and economic reasons have led researchers to develop alternative methods. Among these alternative techniques, the most used is the Limulus amoebocyte lysate (LAL) test [5]. It is also known as bacterial endotoxin test. LAL test can detect or quantify bacterial endotoxins [6].

There are three types of bacterial endotoxin tests: gel-clot technique, which is based on gelation; turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and chromogenic technique, based on the development of colour after cleavage of a synthetic peptide-chromogen [7]. The first type has this advantage: the decision to pass or fail the product under examination is based on the presence or absence of a gel-clot that is visible to the naked eye. For bacterial endotoxin tests, pharmacopoeias [3,7,8] stipulate that the test product must be soluble in or dilutable with water and it is the solution of the test product that is mixed with the LAL reagent. However, some products such as monoolein–water liquid crystalline gels are not soluble in water, and worse, they solidify in contact with water [9,10]. To the best of our knowledge, no pharmacopoeia or other literature has stipulated any protocol for in vitro endotoxin test (LAL test) for water-insoluble gel products.

Since our research group is currently developing monoolein–water liquid crystalline gels of gentamycin for use as bioresorbable implants for the treatment of chronic osteomyelitis [10-12], we set out to develop an in vitro test method for endotoxin in this particular product.

The test product was a monoolein-water gel containing gentamicin (5 %w/w). It was prepared in our laboratory as follows: 10 g of gentamycin sulfate (Id Indis, Aartselar, Belgium) and 160 g of monoolein (Danisco Pharma, Brabrand, Denmark) were separately dissolved in 50 mL of deionized water and 50 mL of ethyl alcohol/ethyl ether 97.1/2.9 (Stella, Liège, Belgium), respectively. The solutions were sterilised by filtration and placed together in a 500 ml sterile glass flask that was then mounted on a rotary evaporator (model R-205, Büchi, Switzerland). The solvent mixture was evaporated at 50 ^oC, at rotating speeds varying from 150 to 235 revolutions per minute (rpm) and pressures ranging from –0.70 to –0.92 bar. The final product was a liquid crystalline gel, which became very viscous in contact with aqueous fluids. The gel was used as a sterile sustained-release implant [10].

The in vitro endotoxins test (LAL test) was performed on samples of three batches of the gels (implants). The following apparatus and reagents were used: heat-stable apparatus (Thermolyne, USA); sterile and apyrogen pipettes; sterile and apyrogen tubes; vial containing 100 IU of standard endotoxins; Limulus amoebocyte lysate (LAL) reagent (Charles River Laboratories, USA) whose labelled lysate sensitivity (l) was 0.015 IU/ml; apyrogen isopropyle myristate; and water for bacterial endotoxin test (Charles River Laboratories, USA). Solutions of standard endotoxins and Limulus amoebocyte lysate were rehydrated with water for bacterial endotoxin test (Charles River Laboratories, USA).

where ∑e = sum of the log end-point concentrations of the dilution series used and f = number of replicates.

The geometric mean end-point concentration is the measured sensitivity of the LAL reagent (IU/ml). If this was not less than 0.5l and not more than 2λ, the labelled sensitivity was confirmed and was used in the subsequent tests performed with this LAL reagent.

As the monoolein–water liquid crystalline gel of gentamicin was insoluble (in water) and became solid in contact with water, we used a technique to extract endotoxin from it as follows. An amount (100 mg) of the gel was placed in a tube and melted at 40 °C in the heat-stage apparatus. The molten gel was dissolved first in 6 ml of isopropyl myristate in the tube, and then 5 ml of water for BET was added. It was mixed for 10 min using a vortex mixer and then left to settle for 20 min. The lipidic phase (the supernatant) was removed and the aqueous phase (extract) used as the test solution. Test for interfering factors was performed to validate this technique of extraction, and to verify the absence of interference of the extract during LAL test.

Solutions A, B, C and D were prepared as shown in Table 1. The extract was the aqueous solution that stemmed from the extraction process. The test was performed as described for confirmation of labelled lysate sensitivity. Different concentrations of solution B were obtained by adding standard endotoxin to the gel so as to give theoretic concentrations of 2λ, 1λ, 0.5λ, and 0.25λ, respectively, in solution B after extraction.

Solution A = solution stemming from endotoxins extraction (from the gel) and being supposed free of detectable endotoxins; Solution B = test for interference; Solution C = control for the labelled lysate sensitivity; Solution D = negative control (water for BET).

The geometric mean end-point concentration of solutions B and C was determined using the expression described for confirmation of the labelled lysate sensitivity. The test was not valid unless all replicates of solutions A and D showed no positive reaction and the results of solution C confirmed the labelled lysate sensitivity. If the sensitivity of the

lysate determined with solution B was not less than 0.5λ and not greater than 2λ, the extract did not contain interfering factors under the experimental conditions and the technique of extraction was validated.

Determination of endotoxin limit concentration in the extract and in the gel

The endotoxin limit concentration (ELC) in the extract was determined according to the procedures of International Pharmacopoeia and European Pharmacopoeia [3,8] using Eq 2.

ELC = (K x C) / M …………………………. (2)

where K = threshold pyrogenic dose of endotoxins per kilogram of body mass in a single hour period; C (concentration of the extract) = sample mass / volume of aqueous phase stemming from extraction; M = maximum recommended dose of product per kilogram of body mass in a single hour period. The endotoxin limit concentration in the gel was calculated as in Eq 3.

ELC_gel = ELC_extract x V/M ……………….. (3)

where V = volume of aqueous phase stemming from extraction) and M = mass of the gel sample.

Determination of maximum valid dilution

Maximum valid dilution (MVD) is the maximum allowable dilution of the extract at which the endotoxin limit can be determined.

MVD = ECL/λ …………………………... (4)

where λ = the labelled lysate sensitivity

Determination of the endotoxins concentration in the gel

Determination of endotoxin concentration in the gels was performed according to European Pharmacopoeia [8], using semi-quantitative test. First, endotoxin concentration of the extract obtained from the gel was determined. Endotoxin concentration in the gel was then computed as in Eq 5.

C_gel = C_extract x V_extract / M_gel …………..... (5)

where C_gel = endotoxin concentration of the gel, C_extract = endotoxin concentration of the

Table 2: Composition of solutions added to LAL reagent at equal volume (100 ml) for the determination of endotoxins concentration in the extract

Solution	Endotoxin concentration/ solution in which endotoxins were added	Diluents	Dilution factor	Endotoxin concentration	Number of replicates
A₁	None/Extract	Water for BET	1 2 4 8		2 2 2 2
B₁	2λ/Extract	Extract	1	2λ	2
C₁	2λ/Water for BET	Water for BET	1 2 4 8	2λ 1λ 0.5λ 0.25λ	2 2 2 2
D₁	None/Water for BET				2

Solution A₁ = solution of endotoxin extract (from gel) and presumed to be free of detectable endotoxins; Solution B₁ = solution A containing standard endotoxin at a concentration of 2l (positive product control); Solution C₁ = series of water for BET containing standard endotoxin at concentrations of 2l, 1l, 0.5l and 0.25l; Solution D₁ = water for BET (negative control)

extract, V_extract = volume of the extract, and M_gel = weigth of the gel sample.

Solutions A₁, B₁, C₁ and D₁ were prepared as shown in Table 2 and tested according to the procedure for the confirmation of the labelled lysate sensitivity described above. The test was not validated unless the following three conditions were met: both replicates of solution D₁ (negative control) were negative; both replicates of solution B₁ (positive product control) were positive; the geometric mean end-point concentration of solution C₁ was in the range of 0.5l to 2l. To determine the endotoxin concentration of solution A₁, the end-point concentration for each replicate series of dilutions was calculated by multiplying each end-point dilution factor by 1l. The endotoxin concentration in the extract was the geometric mean end-point concentration of the replicates (see Eq 1).

If none of the dilutions of the extract was positive, the endotoxins concentration was less than 1l. The gel met the requirements of the BET if its endotoxin concentration was less than the endotoxin limit concentration.

Statistical analysis

Statistical analysis was performed using GraphPad PRISM version 2.01 (GraphPad Software Inc., USA). Values of p < 0.05 were considered significant. Mann-Whitney test was used to compare the endotoxin concentration of the gel and the theoretical (calculated) endotoxin limit concentration (in the gel).

RESULTS

Confirmation of labelled lysate sensitivity

The results of the gel-clot tests to confirm labelled lysate sensitivity are shown in Table 3. Geometric mean end-point = antilog [(3log 0.015 + log 0.0075) / 4] = 0.0126 IU/ml.

Table 3: Results of the gel-clot tests to confirm the labelled lysate sensitivity (n = 4)

Endotoxin concentration	Tube number
Endotoxin concentration	1	2	3	4
2λ (= 0,030 IU/ml)	+	+	+	+
1λ (= 0,015 IU/ml)	+	+	+	+
0.5λ (= 0,0075 IU/ml)	-	-	+	-
0.25λ (= 0,0037 IU/ml)	-	-	-	-
0λ (= water for BET)	-	-	-	-

- = absence of formation of viscous gel (negative reaction)
+ = formation of viscous gel (positive reaction)

Table 4: The results of gel-clot tests to verify absence of interfering factors during BET of the gels (n = 4)

Solution	Endotoxin concentration/ solution in which endotoxins were added	Initial endotoxin concentration	Tube number
Solution		Initial endotoxin concentration	1	2	3	4
A	None/Extract		-	-	-	-
B	2λl/Extract	2λ (= 0,030 IU/ml) 1λ (= 0,015 IU/ml) 0.5λ (= 0,0075 IU/ml) 0.25l (= 0,0037 IU/ml)	+ + - -	+ + - -	+ + - -	+ + - -
C	2λ/Water for BET	2λ 1λ 0.5λ 0.25λ	+ + - -	+ + - -	+ + - -	+ + - -
D	None/Water for BET	0	-	-	-	-

- = absence of formation of viscous gel (negative reaction)
+ = formation of viscous gel (positive reaction)

Therefore, the measured sensitivity of the LAL reagent was 0.0126 IU/ml.

Test for interfering factors

Table 4 shows the results of the gel-clot tests verifying absence of interfering factors during BET of the gel. The geometric mean end-point concentration of solutions B and C (i.e., sensitivity of the lysate with solutions B and C) was equal to antilog [(4log 0.015)/4] = 0,015 IU/ml.

Endotoxin limit concentration (ELC)

The maximum recommended dose of the gel per kg body weight (over a 24 h period) was 1.71 g, i.e., 0.0712 g/kg/h. The lowest threshold pyrogenic dose of endotoxins per kg body weight over a single hour period (K) was 0.2 IU. The concentration of the test solution was 100 mg/5 ml, i.e., 20 mg/ml. ELC in the extract = (0.2 IU/kg/h x 20 mg/ml)/ 71.2 mg/kg/h, i.e., 0.056 IU/ml. Therefore, ELC in the gel was (0.056 IU/ml x 5 ml)/ 100 mg, i.e., 0.0028 IU/mg. Thus, the maximum valid dilution (MVD) of the extract was (0.056 IU/ml)/ 0.015 IU/ml, i.e., 3.73.

Endotoxin concentration in the gel

Table 5 shows the results of gel-clot tests to determine endotoxin concentration in the extract. These results were similar for all the three batches of the gel. The geometric mean end-point concentration of solution C₁ = antilog [(2log 0.015)/2], i.e., 0.015 IU/ml. As neither diluted extract nor initial extract was positive, the endotoxin concentration in the extract was less than l, i.e., 0.015 IU/ml. Therefore, the endotoxin concentration in the

Table 5: The results of gel-clot tests to determine endotoxin concentration in extracts (n = 4)

Solution	Endotoxin concentration/ solution in which endotoxins were added	Dilution factor	Initial endotoxin concentration	Tube
A₁	None/Extract	1 2 4 8		- - - -	- - - -
B₁	2l/Extract	1	2λ	+	+
C₁	2l/Water for BET	1 2 4 8	2λ 1λ 0.5λ 0.25λ	+ + - -	+ + - -
D₁	None/Water for BET		0	-	-

- = absence of formation of viscous gel (negative reaction)
+ = formation of viscous gel (positive reaction)

gel was less than (0.015 IU/ml x 5ml)/100 mg, i.e., 0.00075 IU/mg.

DISCUSSION

The gel-clot technique for bacterial endotoxins test is based on the gelation of a lysate of amoebocytes (limulus amoebocyte lysate) from the horseshoe crab, Limulus polyphemus or Limulus tachypleus. The addition of a solution containing endotoxins (at least 1l concentration) to a solution of the lysate produces gelation of the mixture; l is the labelled lysate sensitivity.

The sensitivity of the lysate (LAL reagent) in the presence or absence of the extract was at least 0.5 times but not more than twice the labelled lysate sensitivity, i.e., between 0.0075 and 0.0300 IU/ml. All negative controls were negative and positive controls were positive for all gel-clot tests (Tables 3 - 5). According to pharmacopoeias [3,7,8], these results confirm the sensitivity of the labelled lysate. The extract neither inhibited nor activated gel formation (see Table 4), suggesting that it was suitable for bacterial endotoxin test. The sensitivity of the lysate remained valid when it was determined with a solution derived from artificially contaminated gel (see results of the test for interfering factors). Therefore, the developed technique achieved the extraction of the whole endotoxin from the gel.

Due to the physicochemical properties of the gel, we could not extract endotoxin directly with water for BET [10]. It was only feasible after dissolution of the gel in lipidic solvent (isopropyl myristate). The hydrophilic property of the endotoxin enabled them to migrate in the aqueous phase after liquid-liquid phase extraction. Endotoxins, which are lipopolysaccharides, are composed of a hydrophilic polysaccharide moiety, which is covalently linked to a hydrophobic lipid moiety [13].

As monoolein–water liquid crystalline gel of gentamicin is a novel product, their endotoxin limit concentration is not specified in the pharmacopoeias used [3,7,8]. However, the pharmacopoeias state recommendations for determining this parameter. In our study, the endotoxin limit concentration was calculated using parameters that afford the greatest safety to patients. Consequently, the lowest threshold pyrogenic dose of endotoxins per kg body weight over a single hour period (K) suggested by European Pharmacopoeia was used (0.2 IU).

The endotoxin concentration in the gels (< 0.00075 IU/mg) was less than the calculated endotoxin limit concentration (0.0028 IU/mg) (p < 0.001). The tested gels were apyrogenic and met the requirements of three pharmacopoeias used [3,7,8]. This assertion is justified by the absence of bacterial endotoxin in the product which implies the absence of pyrogenic components [8]. Though pyrogens are a chemically heterogeneous group of fever-inducing compounds, endotoxins from Gram-negative bacteria are of major concern to the pharmaceutical industry due to their ubiquitous sources, stability and highly toxic reactions [6,14].

CONCLUSION

We have demonstrated that it is possible to perform in vitro endotoxin test (BET) on liquid crystalline gels. Application of this developed technique enabled us to perform in vitro endotoxins test of a monoolein–water liquid crystalline gel intended for use as an implant. The tested batches of gel were apyrogenic under the conditions of the study. The in vitro endotoxin test that we developed may be used to assess the safety of a wide range of insoluble parenteral drugs.

ACKNOWLEDGMENT

The authors would like to thank Coopération Universitaire au Développement/ Commission Interuniversitaire Francophone (CUD/CIUF), Association pour la Promotion de la Formation et de l’Education à l’Etranger (APEFE), and Commissariat Général aux Relations Internationales (CGRI) of Belgium for their financial support for this study.

REFERENCES