# Provocholine (Page 2 of 4)

## 2.5 Reconstitution and Dilution Prior to Administration and Storage of Solutions

Provocholine requires reconsitution before use (see Tables 3 and 4):

- Add 6.25 mL of 0.9% Sodium Chloride Injection (0.9% saline) or 0.9% Sodium Chloride Injection with 0.4% phenol (0.9% saline with 0.4% phenol) to the supplied vials containing 100 mg of Provocholine powder
- Shake the vial to obtain a clear solution

Dilute the reconstituted Provocholine solution:

- Using sterile, empty USP Type 1 borosilicate glass vials, dilute the reconstituted Provocholine solution with 0.9% saline or 0.9% saline with 0.4% phenol either by doubling the concentration (see Table 3) or quadrupling the concentration (see Table 4)
- After adding the diluent, shake each vial to obtain a clear solution
- Use the same diluent to prepare all concentrations

Use a sterile bacterial-retentive filter (porosity 0.22 µm) when transferring the reconstituted or diluted solution from each vial (at least 2 mL) to a nebulizer.

Refrigerate the reconstituted and diluted solutions at 36^{o} to 46^{o} F (2^{o} to 8^{o} C) for up to 2 weeks. Since the temperature of the solution affects nebulizer output, solutions should be taken out of the refrigerator and allowed to equilibrate to room temperature (approximately 30 minutes) before use.

**Table 3: Reconstitution and Dilution of Supplied Provocholine Powder: Doubling Concentrations**

TAKE | ADD 0.9% Saline or 0.9% Saline with 0.4% Phenol | |

100 mg of Provocholine Powder in one supplied vial | 6.25 mL | 16 mg/mL (6.25 mL) (Solution A) |

3 mL of Solution A | 3 mL | 8 mg/mL (6 mL) (Solution B) |

3 mL of Solution B | 3 mL | 4 mg/mL (6 mL) (Solution C) |

3 mL of Solution C | 3 mL | 2 mg/mL (6 mL) (Solution D) |

3 mL of Solution D | 3 mL | 1 mg/mL (6 mL) (Solution E) |

3 mL of Solution E | 3 mL | 0.5 mg/mL (6 mL) (Solution F) |

3 mL of Solution F | 3 mL | 0.25 mg/mL (6 mL) (Solution G) |

3 mL of Solution G | 3 mL | 0.125 mg/mL (6 mL) (Solution H) |

3 mL of Solution H | 3 mL | 0.0625 mg/mL (6 mL) (Solution I) |

**Table 4: Reconstitution and Dilution of Supplied Provocholine Powder: Quadrupling Concentrations**

TAKE | ADD 0.9% Saline or 0.9% Saline with 0.4% Phenol | Concentration (Total Volume) after reconstitution or dilution |

100 mg of Provocholine Powder in one supplied vial | 6.25 mL | 16 mg/mL (6.25 mL) (Solution 1) |

3 mL of Solution 1 | 9 mL | 4 mg/mL (12 mL) (Solution 2) |

3 mL of Solution 2 | 9 mL | 1 mg/mL (12 mL) (Solution 3) |

3 mL of Solution 3 | 9 mL | 0.25 mg/mL (12 mL) (Solution 4) |

3 mL of Solution 4 | 9 mL | 0.0625 mg/mL (12 mL) (Solution 5) |

## 2.6 Calculation and Interpretation of Methacholine Challenge Test Results

A positive methacholine challenge test is a ≥ 20% reduction in the FEV_{1} (after Provocholine oral inhalation) compared with the mean post-diluent FEV_{1} . Calculate and record post-diluent FEV_{1} value before the methacholine challenge test is started. Express airway hyperreactivity as the provocative Provocholine concentration (mg/mL) providing a fall in FEV_{1} of ≥ 20% (PC_{20} ) when the methacholine challenge test is dosed using either the 5-breath dosimeter method or the 2-minute tidal breathing method, or as the provocative Provocholine dose (mcg) providing a fall in FEV_{1} of ≥ 20% (PD_{20} ) when using the 2-minute tidal breathing method.

Calculation of PC_{20}

Calculate PC_{20} using one of the following methods. Determine the percent decrease in FEV_{1} using the mean post-diluent FEV_{1} and the lowest FEV_{1} post-dose, as shown below:

% fall in FEV_{1} = mean post-diluent FEV_{1} — lowest FEV_{1} post-Provocholine x 100

mean post-diluent FEV_{1}

Method #1

Plot the percent decrease in FEV_{1} against the increasing methacholine concentration using a log scale and obtain the PC_{20} by linear interpolation between the last two points, as shown in Figure 1.

Method #2

Alternatively, calculate the PC_{20} as follows:

PC_{20} = antilog [log C1+ (log C2 -- log C1)(20 -- R1)]

(R2- R1)

Where:

• C1 = second last methacholine concentration (< 20% FEV_{1} decrease)

• C2 = last methacholine concentration (≥ 20% FEV_{1} decrease)

• R1 = % fall FEV_{1} after C1

• R2 = % fall FEV_{1} after C2

Calculation of PD_{20} (2-minute tidal breathing method only)

Calculate the PD_{20} as follows:

PD_{20} = antilog [log D1+ (log D2 -- log D1)(20 -- R1)]

(R2- R1)

Where:

• D1 = second last Provocholine dose (< 20% FEV_{1} decrease)

• D2 = last Provocholine dose (≥ 20% FEV_{1} decrease)

• R1 = % FEV_{1} decrease after D1

• R2 = % FEV_{1} decrease after D2

When using the English Wright nebulizer, refer to Table 2 for D1 and D2.

Interpretation of Results

A negative (normal) methacholine challenge result is defined as FEV_{1} reduction of < 20% after all the doses (doubling or quadrupling dose increments) in Table 1 (for 5-breath dosimeter method) or Table 2 (for the 2-minute tidal breathing method) have been administered.

If asthma drugs are discontinued prior to the methacholine challenge test consider the possibility of rebound airway hyperreactivity in the interpretation of the test results. The methacholine challenge test may occasionally be falsely positive after an influenza infection or upper respiratory infection, immunizations, in very young or very old patients, in patients with chronic lung disease (e.g., cystic fibrosis, sarcoidosis, tuberculosis, chronic obstructive pulmonary disease), in patients with allergic rhinitis without asthma symptoms, in smokers, or in patients after exposure to air pollutants.

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